Shark cartilage extract: process of making, methods of using, and compositions thereof

ABSTRACT

The present invention relates to cartilage extracts and to a method of producing the same. Shark cartilage extracts having anti-angiogenic, anti-tumor, anti-inflammatory and anti-collagenolytic activities have been obtained by an improved process. The process comprises the steps of obtaining a crude cartilage extract in an aqueous solution, this crude extract being fractionated to recover molecules of a molecular weight less than about 500 kDa. Some of the biologically active components of the extract are prepared by further fractionation. The cartilage extract can be used for treating diseases or conditions having etiological components selected from the group consisting of tumor proliferation, angiogenesis, inflammation, metalloprotease activity and collagenolysis. Several cosmetic applications based on the capacity of the liquid extract to improve skin conditions are also disclosed. A simple and efficient process for the preparation of cartilage extracts is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser.No. 09/504,065, filed Feb. 15, 2000; which is a continuation-in-part ofU.S. patent application Ser. No. 08/693,535, filed Aug. 8, 1996; whichis in turn a continuation-in-part of U.S. patent application Ser. No.08/550,003, filed Oct. 30, 1995; which is in turn a continuation-in-partof U.S. patent application Ser. No. 08/384,555, filed Feb. 3, 1995, nowU.S. Pat. No. 5,618,925; which is a continuation-in-part of U.S. patentapplication Ser. No. 08/234,019, filed on Apr. 28, 1994, now abandoned.

BACKGROUND OF THE INVENTION

[0002] Cartilage is a/n avascularized tissue and has been studied as apotential candidate containing anti-angiogenic factors. It is also atissue that is relatively resistant to tumor development. The tumorassociated with cartilage, chondrosarcoma, is the least vascularized ofsolid tumors. Angiogenesis is one of the important factors in thedevelopment of a tumor. Discrete solid tumor masses appear if the tumorcells can provoke the adjacent vascular network to expand in order tosupply their nutritional needs. The factors involved in the stimulationof angiogenesis have been studied for their role in the development oftumor and anti-angiogenic factors. Drugs having an angiogenic inhibitoryactivity have been also investigated as tools for controlling the growthor for effecting regression of tumors.

[0003] It has been discovered that scapular cartilage in calves containsa substance that inhibits the vascularization of solid tumors (Langer etal. (1976) Science. 193: 70-72). Because of its encouraging potential asanti-tumor agent, sources of greater supplier of cartilage have beenlooked for.

[0004] Sharks are a potential source of this kind of angiogenesisinhibitor because their endoskeleton is composed entirely of cartilage(6% of their body weight versus 0.6% in calves). Sharks have a lowpropensity for tumor development. Many hypotheses have been elaboratedto explain this low probability of developing tumors in sharks. It hasbeen shown inter alia that IgM antibodies able to readily attack anyaggressing agent, or that macrophages capable of differentiating normalcells from neoplastic cells and of destroying the latter. Rosen andWoodhead (1980) in Medical Hypotheses. 6:441-446 have postulated thatthe rarity of tumors in elasmobranchs (a subclass to which sharks andrays belong) might be due to the high ionic strength of their tissues,which is equivalent to a high body temperature. In these conditions,these authors believe that the immune system exerts a close to 100%immunological surveillance. It has also been discovered that sharksproduce an aminosterol having antibacterial and antiprotozoalproperties.

[0005] Finally, Lee and Langer (1983) in Science 221: 1185-1187 andFolkman and Klagsbrun (1987) in Science. 235: 442-447 have shown thatsharks produce a substance that inhibits neovascularization. Lee andLanger (op. cit.) have isolated this substance by extracting it fromshark cartilage in denaturing conditions (guanidine extraction). Thisprocess of extraction is however very long (41 days), and the yield ofactive components is far from excellent. While the active substanceisolated from calves has a molecular weight of about 16 kilodaltons(kDa), the same group of researchers has not given a precise molecularweight to the one isolated from sharks. This substance is only definedhas having a molecular weight higher than 3500 Daltons.

[0006] Oikawa et al. (1990) in A Novel Angiogenic Inhibitor Derived fromJapanese Shark Cartilage (I). Extraction and Estimation of InhibitoryActivities Toward Tumor and Embryonic Angiogenesis (Cancer Letters 51:181-186) have applied the same method of extraction as the one describedby Lee and Langer, but of a much shorter duration (2 days instead of 41days). The anti-angiogenic substance isolated from shark cartilage byOikawa et al. is restricted to a molecule having a molecular weightranging from 1000 to 10,000 Daltons. Schinitsky (U.S. Pat. No.4,473,551) has described a water extract of crude powdered sharkcartilage which fraction of more than 100,000 Daltons has ananti-inflammatory activity, especially in combination with glucosamine.No suggestion of a component of this extract having an anti-angiogenicor anti-tumor activity is made in this patent.

[0007] Kuetner et al. (U.S. Pat. No. 4,746,729) have isolated aolymorphonuclear neutrophil (PMN) elastase inhibitor from bovinecartilage. This inhibitor has been obtained from an aqueous extract ofcartilage from which molecules of a molecular weight of less than 50,000Daltons have been retained. Fractionation on Sephacryl S-200 has givennumerous fractions from which those of 10-40 kDa have been pooled afterthey have demonstrated an anti-elastase activity. The active componenthas an isoelectric point of 9.5 and might have a molecular weight ofabout 15,000 Daltons. Kuetner et al. (U.S. Pat. No. 4,042,457) have alsoshown that bovine cartilage has a component of a molecular weight ofless than 50,000 Daltons which has a cell proliferation inhibitoryactivity without any activity on endothelial cell growth. Balassa et al.(U.S. Pat. No. 4, 822, 607) have obtained a cartilage extract in anaqueous solution, which extract has an anti-tumor activity. However, wehave observed no anti-angiogenic activity in an extract obtained byreproducing Balassa's method. Spilburg et al. (U.S. Pat. No. 4,243,582)have isolated two glycoproteins of molecular weight of 65 kDa and ofisoelectric point of 3.8 from bovine cartilage (guanidine-extraction)which show anti-trypsin activity and an endothelial cell growthinhibitory activity.

[0008] Calf and shark cartilage contain many biological activities suchas pro-inflammatory activity, anti-inflammatory activity,anti-angiogenic activity, lysozyme activity, cell growth-promotingactivity, inhibitory activity against types I and IV collagenase,elastase, and other proteases like trypsin, chymotrypsin and plasmin.However, nobody has yet obtained a cartilage extract that comprises apool of clinically valuable activities, and particularly with newactivities.

[0009] Shark cartilage anti-angiogenic component(s) have been generallytested in rabbit corneal pocket assay or in chick chorioallantoicmembrane (CAM) assay. Up to date, whole powdered cartilage has beentested directly on tumors in vivo, on human melanoma xenograft implantedin nude mice (U.S. Pat. No. 5,075,112), as well as tested in CAM testsfor its anti-angiogenic effect. Even though an anti-tumor effect hasbeen assigned to cartilage extracts, this effect has most often beenattributed to the anti-angiogenic component that deprives the tumor ofblood supply. Up to now, there is no evidence in the art that a sharkcartilage has a direct effect on tumor cell proliferation.

[0010] A few methods of obtaining shark cartilage extracts and fractionsare already known. Some of them produce a powdered crude cartilagewithout any extraction (U.S. Pat. No. 5,075,112). Others use denaturingor chaotropic agents like guanidine (U.S. Pat. No. 4,243,582). Othersperform a pre-treatment of cartilage by way of an enzymatic digestion toget rid of any muscular, nervous or vascular structures surrounding thecartilage, which pre-treatment step is followed by the elimination offats in organic solvents, and then the active components are extractedin an aqueous phase. (Balassa et al., U.S. Pat. Nos. 3,478,146;4,350,682; 4,656,137; and 4,822,607). The effect of such pre-treatmenton the preservation of the integrity of the biologically activecartilage components is not known. If too extensive, an enzyme digestionmay hydrolyze active protein components. For example, Balassa's method(U.S. Pat. No. 4,822,607) produces a liquid extract withoutanti-angiogenic activity; this loss may be the result of such enzymaticdegradation, or else, the anti-angiogenic is masked or antagonized byother molecules. Balassa's method does not include a fractionation stepwhich would further enrich an extract in active components, and whichmay remove undesirable molecules. Others simply produce aqueous extracts(in water (U.S. Pat. No. 4,473,551) or salt solutions (U.S. Pat. No.4,746,729) of cartilage by eliminating the unsolubilized material. Amongthe latter, specific fractions of specific molecular weights have beenparticularly retained for further study and purification (see discussionabove). There is no process in the art that leads to the preparation ofa cartilage extract having substantially all the hydrosoluble activecomponents of cartilage.

[0011] Moreover, the methods of the prior art are too lengthy to be of apractical purpose and they do not necessarily yield sufficient amountsof active components. Amongst the recovered components, some are notrecovered at all or in insufficient yield to show detectable activity orsome have been disregarded by focusing on the preparation of onespecific component having one activity.

[0012] Angiogenesis is not only involved in cancer development. Manydiseases or conditions affecting different physiological systems(indicated in parentheses) are angiogenesis-dependent among which thefollowing examples: arthritis and atherosclerotic plaques (bone andligaments), diabetic retinopathy, neovascular glaucoma, maculardegeneration, ocular herpes, trachoma and corneal graftneovascularization (eye), psoriasis, scleroderma, rosacea, hemangiomaand hypertrophic scarring (skin), vascular adhesions and angiofibroma(blood system). Therefore, any new and potent anti-angiogenic “factor”could find an use in the treatment of these diseases as well as incancer therapy and other angiogeno-dependent diseases. Moreover, sincemany of the above-mentioned diseases and conditions also have aninflammatory etiological component, any new and potent anti-inflammatory“factor” or component could find a use in the treatment of thesediseases and conditions as well as of any other inflammatory diseases orconditions. Furthermore, since metalloproteases like collagenases areinvolved in a diversity of diseases and conditions like cancer,inflammation and premature ageing of the skin (collagen degradingactivity), a new and potent anti-collagenolytic “factor” could be usefulin the treatment of diseases or conditions having a collagenolytic or amatrix metalloprotease etiological component. Because angiogenesis,inflammation and proteolysis may be encountered alone or in combinationin a large variety of diseases or conditions, a product capable ofantagonizing at least all these activities without affecting normal bodyfunctions would be of a great therapeutic value. Furthermore, a productwhich would have a direct anti-tumor activity would also have asignificant therapeutic value.

BRIEF SUMMARY OF THE INVENTION

[0013] The present invention provides a new method of producingcartilage extracts which has the advantage of containing a plurality oftherapeutically valuable activities. Amongst those, anti-angiogenic,anti-inflammatory, anti-collagenolytic, in vivo anti-tumor proliferatingand direct in vitro anti-tumor proliferating activities have beenconfirmed to be present in satisfying concentrations in a cartilageextract obtained from shark. All activities have been obtained in aliquid extract of shark cartilage, and some of them have been obtainedor verified as being present in a solid extract of the same.

[0014] The present invention relates to a new method of preparing afractionated cartilage extract comprising water soluble biologicallyactive components, the majority of which have a molecular weight of lessthan about 500 kDa. The method comprises the step of:

[0015] first fractionating a crude cartilage extract comprisingwater-soluble biologically active components obtained from cartilagematerial, such that a major portion of the biologically activecomponents having a molecular weight of greater than about 500 kDa areseparated from a major portion of biologically active components havinga molecular weight of less than about 500 kDa to form a firstfractionated cartilage extract.

[0016] A first fractionated cartilage extract that has been obtainedfrom shark cartilage is referred to herein below as a “cartilageextract.” The term fractionated will be omitted, even though thecartilage extract has been fractionated to remove molecules heavier thanabout 500 kDa.

[0017] This method has the advantage of being easy to perform andefficient. High yields of cartilage extract have been obtained, whichextract, particularly obtained from fresh or frozen/thawed sharkcartilage, contains at least all the above-mentioned biologicalactivities. Other sources of cartilage can be used in this process. Itis preferably performed at cold, cool or ambient temperatures (about 0°to 20° C., although the biologically active components can withstandtemperatures as high as about 40° C.), in non-denaturing conditions(preferably, the extraction medium is an aqueous solution or purewater), at a near neutral pH (about 5 to 8) to maximize the probabilityof recovering compounds of a priori unknown physio-chemicalcharacteristics.

[0018] According to this process, cartilage components can be extractedin a low volume of solution (as low as 1 L for 1 kg of cartilage).Homogenization of cartilage may be performed for a short period of time(as short as 10 to 20 minutes). Homogenization and extraction in theextraction medium results in the formation of particles and a crudeextract which are separated by mechanical and physical means. The crudeextract is then fractionated to remove molecules having a molecularweight higher than 500 kDa. A liquid extract is the fractionated extractcomprising water-soluble biologically active components having amolecular weight less than about 500 kDa. In one embodiment, the solidextract comprises the insoluble particles separated from the liquidextract. In another embodiment, the solid extract is a dried form of theliquid extract. Homogenization reduces the size of cartilage particlesand thereby maximizes the extractable surface area of the cartilage.Other known ways of reducing the particle size of solids, in particularcartilage, may be used, as long as a major portion of the biologicallyactive components being extracted are preserved and remain extractable.

[0019] This invention relates to cartilage extracts, particularly toextracts from elasmobranch species, more particularly shark. The solidextract has shown activity. It may contain collagen and non-hydrosolublecomponents. It may also contain a residual activity of what wasextracted in the cartilage extract. The cartilage extract is very richin activities. It can be used as such or it can be concentrated. Aconcentration step which favors the maintenance of biological activitiesis conducted. Ultrafiltration on a membrane having a nominal molecularweight cut-off value of about 1 kDa has been used to concentrate thecartilage extract of this invention. Nanofiltration on a membrane havinga nominal molecular weight cut-off value of about 100 Daltons shouldeven be better to concentrate the biological activities of the cartilageextract, preventing the loss of any activity assigned to very smallmolecules. Finally, concentration by evaporation or lyophilization canalso be performed, in so far as such treatment does not substantiallyreduce the activity of the extract. Stabilizers may be used to protectthe cartilage extract to minimize loss of activity duringlyophilization.

[0020] The cartilage extract (<500 kDa) has been further fractionated tocharacterize the active components thereof. Active fractions have beenobtained by performing additional fractionation steps. Some fractionstested for their anti-tumor activity on tumor cell lines have beengrossly characterized by their molecular weight and isoelectric point.Others have been assigned an activity, particularly anti-collagenolytic,anti-metalloproteasic or anti-angiogenic. Therefore, valuable activitiesare recovered in cartilage extract and fractions thereof, which may beadvantageously used. In lieu of administering high amounts of powderedcartilage, a more acceptable and enriched extract may now beadministered.

[0021] The present invention also relates to any therapeutic or cosmeticcompositions comprising as an active ingredient an effective amount of aconcentrated or dilute cartilage extract preferably obtained from shark.These compositions are generally used in dermatological or cosmeticformulations due to the observed activities of the cartilage extract. Inthis respect, the observed anti-angiogenic, anti-metalloproteasic andanti-inflammatory activities, and the antagonistic effect of cellulardifferentiation mediated by the protein kinase C signal transductionpathways in keratinocytes, and the antagonistic effect on VEGF (vascularendothelium growth factor) activity, are all considered as possiblemechanisms upon which new uses of the shark cartilage extract can bedeveloped.

[0022] The invention also provides a method of treating a variety ofmammalian skin diseases or disorders including, for example, Reiter'ssyndrome, pityriasis rosea, lichen planus, pityriasis rubra pilaris,secondary syphilis, mycosis fungoides, ichthyosiform eruptions,sclerodermia, hyperthrophic scar, papulosquamous disease, psoriasis,rosacea, eczema and acne. Preferred embodiments of the invention providethe following:

[0023] 1) a method for inhibiting angiogenesis in mammalian skin;

[0024] 2) a method for reducing telangiectesia in mammalian skin;

[0025] 3) a method for reducing spider veins or varicose veins inmammalian skin;

[0026] 4) a method for inhibiting endothelial cell proliferation inmammalian skin;

[0027] 5) a method for treating cancer in mammalian skin, particularly amelanoma;

[0028] 6) a method for decreasing the expression of rosacea in mammalianskin;

[0029] 7) a method for reducing papulosquamous skin disease inmammalian;

[0030] 8) a method for reducing the appearance of peri-orbital darkcircles in mammalian;

[0031] 9) a method for reducing inflammation in mammalian, be it causedby a chemical irritant, a physical abrasion, U.V. radiation, an allergenor an infectious agent;

[0032] 10) a method for soothing irritated skin in mammalian;

[0033] 11) a method for inhibiting activated-keratinocytedifferentiation in mammalian skin;

[0034] 12) a method for reducing acne in mammalian skin;

[0035] 13) a method for decreasing the expression of eczema in mammalianskin;

[0036] 14) a method for inhibiting metalloprotease activity in mammalianskin;

[0037] 15) a method for treating warts in mammalian;

[0038] 16) a method for enhancing wound repair in mammalian;

[0039] 17) a method for enhancing skin barrier function in mammalian;

[0040] 18) a method for regulating wrinkles and atrophy in mammalianskin; and

[0041] 19) a method for retarding premature aging in mammalian skin.

[0042] A plurality of diseases or disorders having simple or complexetiologies will benefit from treatment with the present cartilageextract. Other medical fields of use, including but not limited todermatological use, are within the scope of this invention.Dermatological diseases or disorders that have up to date been difficultto treat with actual short and long term therapies because of theircomplex etiology, are susceptible to treatment with the cartilageextract of this invention. Furthermore, since the cartilage extract hasbeen successfully tested in a variety of cancers, arthritis, psoriasis,rosacea, and acne cases, compositions and methods for treating diseases,conditions or disorders having one or more etiological componentsselected from tumor proliferation, angiogenesis, inflammation andmetalloprotease activity such as collagenolysis, are within the scope ofthis invention, without any limitation as to the dosage form, the routeof administration way and the tissue to be treated.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0043] The present invention will be more readily understood by way ofthe specific embodiments shown in the appended figures which variousembodiments of the invention.

[0044] In the drawings:

[0045]FIG. 1 shows the specific amino acid composition of the cartilageextract (liquid).

[0046]FIG. 2 shows a dose-response inhibitory activity of sharkcartilage (solid) extract on ZR75-1 and MCF-7 cell lines.

[0047]FIG. 3 illustrates dose-response curves of MCF-7 cells in thepresence of increasing concentrations of estradiol with or without twoconcentrations of solid cartilage extract.

[0048]FIGS. 4a) and b) show a comparison of mammary gland tumor sectionsof rats which have been administered by gavage of water (A) or acombination of solid and liquid cartilage extracts (B).

[0049]FIG. 5 show that the vascularization of the mammary gland andtumor in cartilage-treated rats is decreased by 50%.

[0050]FIG. 6 shows that the liquid cartilage extract has no effect onfibroblast cell proliferation.

[0051]FIG. 7 shows a dose-response curve inhibition of liquid cartilageextract on HUVECs proliferation.

[0052]FIG. 8 shows that liquid cartilage extract inhibits TPA-inducedkeratinocyte differentiation.

[0053]FIG. 9 shows a dose-response curve inhibition of liquid cartilageextract on collagenase activity.

[0054]FIG. 10 shows a dose-response curve inhibition of liquid cartilageextract on the Embryonic Vascularization Test (ex ovo).

[0055]FIG. 11 shows the effect of various doses of liquid cartilageextract on tumor growth inhibition in mice.

[0056]FIG. 12 shows that intraperitoneal administration of the liquidcartilage extract can significantly increase the efficacy of liquidcartilage to inhibit tumor growth.

[0057]FIG. 13 represents the electrophoretic profile in non-denaturingconditions of liquid fractions separated on Rotofor; molecular weightmarkers appear at the left followed by a sample of liquid extract beforefractionation, for comparison with the isolated fractions.

[0058]FIG. 14 shows a HPLC migration pattern of a fraction of the liquidextract of this invention having molecular weight lower than 10,000Daltons, which fraction has been concentrated and separated in fivesub-fractions.

[0059]FIG. 15 shows the EVT results obtained with two fractions ofliquid extract of shark cartilage of our invention (DUP), one havingmolecular weight lower than 10,000 Daltons, the other one havingmolecules higher than 10,000 Daltons.

[0060]FIG. 16 shows a FPLC migration pattern of three different extractsof shark cartilage. In panel A, DUP stands for a liquid cartilageextract according to this invention. In panels B and C, BAL and OIKstand for extracts of the prior art, Balassa et al. and Oikawa et al.,respectively.

[0061]FIG. 17 shows a HPLC migration pattern of the same extractsdefined in FIG. 16.

[0062]FIG. 18 shows a CZE comparison of the liquid cartilage extract ofthe present invention with the prior art.

[0063]FIG. 19 shows an EVT comparison of the liquid cartilage extract ofthe present invention with the prior art.

[0064]FIG. 20 illustrates a comparison of the amino acid content of theliquid cartilage extract of the present invention with the prior art.

[0065]FIG. 21 illustrates the significant improvement of the conditionof two patients suffering of psoriasis, one with hyperkeratosis 22 a)and b), and the other one without hyperkeratosis 22 c) and d), whentreated with a topical composition containing an effective amount ofconcentrated liquid cartilage extract (lower photographs) compared withtheir initial condition (upper photographs).

[0066]FIG. 22 shows the improvement of the appearance of spider veins inthe visage of humans treated with liquid cartilage extract.

[0067]FIG. 23 shows the improvement of the appearance of dark circlesaround the eyes of humans treated with liquid cartilage extract.

[0068]FIG. 24 shows the improvement of the appearance of varicose veinson the legs of humans treated with liquid cartilage extract.

[0069]FIG. 25 illustrates the significant improvement of the conditionof a patient suffering of acne when treated with a topical compositioncontaining an effective amount of liquid cartilage extract (lowerphotograph) compared with her initial condition (upper photograph).

[0070]FIG. 26 shows the anti-inflammatory potential of the liquidcartilage extract on human skin.

[0071]FIG. 27 shows the improvement of the barrier function of the skinof humans treated with liquid cartilage extract.

[0072]FIG. 28a) shows the effect of VEGF on HUVEC proliferation, and

[0073]FIG. 28b) shows that the present cartilage extract counteractsVEGF activity on HUVEC proliferation.

[0074]FIG. 29a) shows that the present cartilage extract interferes insome way with the binding of VEGF to its receptor in HUVEC and BEACmodels. This effect is specific to VEGF since (FIG. 29b)) shows that nosuch competition is observed against the binding of bFGF to itsreceptor.

[0075]FIG. 30 shows that the present cartilage extract inhibits theextravasation induced by VEGF. This is presumed to be one of the ways bywhich the extract hinders the initiation of edema and inflammation atthe origin of a cascade of events involved in extracellular matrixremodelling.

[0076]FIG. 31 shows that at least a part of the interference of thecartilage extract with VEGF activity is due to an inhibition of thephosphorylation of VEGF receptor.

[0077]FIG. 32 shows an immunoblot of the cartilage extract revealed withanti-TIMP antibodies. Five μg of TIMP-1, TIMP-2 and TIMP-3 proteins and35 μg of the liquid cartilage extract proteins were subjected toSDS-PAGE and blotted using respective specific anti-TIMP antibodiescoupled to horseradish peroxidase.

DETAILED DESCRIPTION OF THE INVENTION

[0078] In a specific embodiment, cartilage has been obtained fromhealthy small coastal sharks (Dog Fish, smooth hound). Any muscular andconnective tissue has been removed by scraping with ethanol-treatedscalpels and scissors. The cartilage was then vacuum-packed in plasticbags and frozen to −20° C. for further use. Any source of cartilage maybe used in the present invention. It is believed that cartilage from anyelasmobranch species (which includes sharks and rays as species of thissubclass), will provide about equivalent products. The cartilage extractprepared herein may differ in its nature and concentration ofbiologically active components obtained by the present process whencompared to an extract of a mammalian source of cartilage.

[0079] Any variation in the preparation of cartilage prior to itsextraction may be used as long as it does not substantially affect theactivity of the product of interest (a cartilage extract or a particularsub-fraction thereof, for example). Some active components may beresistant to the proteolytic digestion used by Balassa et al. (U.S. Pat.No. 4,822,607) to rid the cartilage of surrounding tissues, while othersmay not be stable to such treatment. Therefore, if one wants to producea liquid extract containing as much as possible of all the hydrosolubleactive components described herein, such a digestion step during theextraction procedure should be avoided or carefully monitored to preventextensive hydrolysis or proteolysis.

[0080] Preparation of Cartilage Extracts

[0081] Clean cartilage was used fresh, thawed to 4° C., or frozen.Cartilage was then passed numerous times (more particularly three times)through the pores of an ethanol-aseptized meat chopper together with anadequate volume of water (an equal quantity (weight/volume) is about aminimal volume but can be increased without bearing any deleteriouseffect on the yield of recovery of valuable components). A low volume ispreferred since it is more convenient to manipulate than unnecessaryhigh volumes, from a practical point of view. In the practice, water hasbeen purified by inverse osmosis and multiple filtrations down to 0.1 μmfilter. Many aqueous solutions (containing salts, for example) could beused in lieu of water. When recovery of a plurality of hydrosolubleactivities is contemplated, working at a near neutral pH (5.0 to 8.0)and non-denaturing conditions is preferred to avoid lysis ordenaturation of some of the cartilage active components. The behavior ofunknown proteins in aqueous solvents is not predictable; some may bemore stable in an acidic pH, some at a basic pH. Furthermore, someproteins may be extractable in mild denaturing conditions, if suchdenaturation does not irreversibly affect the renaturation of theseproteins in aqueous solutions. For the sake of clarity, any extractionmedium that is compatible with the preservation of biologically activehydrosoluble cartilage component is within the scope of this invention.Therefore, performing the extraction in pure water is preferred. Otherpreferred embodiments include those wherein salts and/or chaotropicagents are added to the water prior to or during extraction.

[0082] The cartilage/water mixture was then homogenized at maximum speedin a kitchen blender at about 4 to 20° C. for about 20 minutes. Duringhomogenization, the temperature may be as high as about 40° C. The speedof the agitation as well as the volume of aqueous solution may influenceboth time and yield of extraction. The homogenization (to yield particlesize of less than 500 μm particles) may last from 10 minutes to as 24hours, preferably between about 10 and 60 minutes.

[0083] Liquefaction of this homogenate is performed by disintegrationwith a Polytron disintegrator for about 10 minutes at about 4° C. to 20°C., if the blender did not sufficiently reduce the size of theparticles. Alternatively, the cartilage/water mixture is simplyhomogenized in a higher performance blender-disintegrator which, in ourhands, saved 10 min in the liquefaction step. Upon completion of thehomogenization step, the average residual particle size is preferablyless than about 500 μm. The size of the particles after homogenizationdoes not need to be ultra small, although a very small size shouldincrease extraction efficiency. Indeed, pulverization of cartilage inthe form of a powder before aqueous extraction may be used to reducesize particles prior to extraction, in so far as this does not denaturevaluable activities. In our process, homogenization of cartilage allowssize reduction and extraction of active components to occur almostsimultaneously.

[0084] The homogenate, which is a mixture of particles and of a crudeextract, was centrifuged at 13,600× g during 15 minutes at about 4° C.to 20° C., which step is one way to separate quickly and efficiently asupernatant from a pellet. Variation and adjustment of these parametersare well within the knowledge of the skilled artisan, merely dependingon the volume of homogenate and of the equipment used.

[0085] The resulting pellet was lyophilized for 24 to 48 hours. Thisfirst fraction will hereinbelow be defined as the lyophilizate or “solidextract.”

[0086] The supernatant can be filtered on a 24 μm Whatman filter, ifnecessary, to get rid of particles susceptible to affect the performanceof an ultrafiltration column. The filtered material was then separatedinto fractions. Separation may be achieved by different methods andseparation media including but not limited to:

[0087] Chromatography: (adsorption, ionic exchange, gel filtration)

[0088] Electrophoresis

[0089] Ultrafiltration

[0090] Ultra centrifugation with zonal density gradients, and

[0091] Adsorption extraction.

[0092] In a specific embodiment, the crude extract was ultrafiltered atabout 4° C. to 20° C. (although temperature could increase to about 40°C.) on a tangential flow filtration column having a membrane of aporosity of about 500 kDa, which allowed a first fractionated extract tobe obtained, comprising hydrosoluble molecules of a molecular weightcomprised between 0 and about 500 kDa. This fractionated extract wasfiltered on 0.22 μm filter, and aliquoted in aseptic bottles for furtheruse. This fraction will be further referred to as the “firstfractionated extract” or “cartilage extract” or the “liquid extract.”

[0093] An alternative, higher performing centrifuging procedure has beendeveloped to obtain the pellet and the supernatant. The step ofcentrifuging at 13600× g for 15 minutes followed by a gross filtrationon Whatman filters has been replaced by a centrifugation in a CEPAcentrifuge equipped with a nylon pocket of a porosity of 1 μm, at3000-4000× g. A 25 kg/25 L preparation can be centrifuged in that mannerwithin 30 minutes and provide about 29 liters of supernatant. Theaqueous volume obtained is higher than the starting volume of water,suggesting that a part of the water content of the cartilage itself hasbeen harvested. The solids recovered upon centrifugation and referred toas the “solid extract” were optionally lyophilized. In one example, thesolid and liquid extracts had the following approximate compositionwhich grossly takes into account the variations observed from batch tobatch, and when using different material: Solid Extract: Lipids 7.35%¹Proteins 46.2%² Humidity 20.4% Sodium 4.16 mg/g³ Potassium 2.64 mg/gCalcium 114 mg/g Magnesium 1.49 mg/g Zinc and iron traces LiquidExtract: Lipids 0.10-0.20%¹ Proteins 8-25 mg/ml² Dry weight 8-25 mg/mlHumidity 97-99% Sodium 30-220 mg/100 g³ Potassium 30-40 mg/100 g Calcium2.0 mg/100 g Magnesium 1.1 mg/100 g Zinc and iron traces

[0094] The protein content is evaluated by the Kjeldahl method, whichindeed measures organic nitrogen (N). Organic nitrogen is converted toequivalent protein by using the following equation:${{Proteic}\quad {{content}{\quad \quad}\left( {{mg}/{ml}} \right)}} = \frac{\left( {\% \quad N \times 6.25} \right)}{100}$

[0095] Carbohydrate content was not determined, but carbohydrates wereassumed to be present in the form of proteoglycans and/ormucopolysaccharides. It is possible that these compounds are included inthe measured level of humidity. The solid extract contains an unexpectedlevel of humidity which was measured by the OH groups to be about 20%water content, which is close to the percentage of carbohydratesnormally retrieved in cartilage.

[0096] The (liquid) cartilage extract was analyzed for its amino acidcontent. The average amount of total amino acids was approximately 1.1mg per ml, with the free amino acids accounting for 0.67 mg (61%) andthe amino acids of protein origin accounting for 0.44 mg (39%). Thedistribution of each amino acid is shown in FIG. 1. Significant amountof taurine was also detected (not shown).

[0097] The major amino acids present in the (liquid) cartilage extractare representative of proteins and peptides from cartilage. For example,lysine, glycine, aspartic acid and glutamic acid represent a largeproportion of the amino acid content of the liquid extract and are themain components of the N-telopeptide intermolecular cross-linking incollagen (Hanson et al. (1992) J. Bone & Min. Res. 7: 1251-1258).

[0098] Microbial limit of the liquid extract has been controlled,applying USP XXIII <61> standards.

[0099] Activity Assays

[0100] Solid Extract:

[0101] In vitro Assays:

[0102] These assays have been conducted on the hormone-dependent cancercell lines MCF-7 and ZR75-1 (ATCC (R) numbers 22-HTB and 1500-CRL,respectively).

[0103] ZR75-1 Cells:

[0104] Basal RPMI Medium:

[0105] 52 g RPMI 1640 without phenol red (Sigma R8755), 17.875 g Hepes(free acid; Sigma H0763), 0.55 g sodium pyruvate (Sigma P5280) and 10 gNaHCO₃ were mixed in 5 L of pure water and made pH 7.40 with NaOH.

[0106] If not used immediately, this solution must be protected fromlight to preserve photolabile substances. This solution was filtered,distributed in 500 mL sterile bottles and stored at 4° C. for a maximalperiod of three months.

[0107] Cell Culture Maintenance Medium:

[0108] Basal RPMI medium was supplemented with 10% (v/v) FBS (fetalbovine serum), 100 U penicillin G/50 μg streptomycin sulfate (SigmaP0906)/mL medium, 2 mM L-glutamine (Sigma G1517) and 1 nM E₂(β-estradiol Sigma E8875).

[0109] Experimental Medium:

[0110] Basal RPMI medium was supplemented with 5% FBSA (fetal bovineserum adsorbed on dextran-charcoal), 2 mM L-Glutamine, 100 U penicillinG/50 μg streptomycin sulfate/mL medium and 50 ng/mL insulin (Sigma). Tothis medium was added increasing concentrations of the above-describedsolid extract as well as different concentrations of E₂ (10⁻¹² to ⁻⁵ M).

[0111] MCF-7 Cells:

[0112] BASAL DME-F12 Medium:

[0113] DME-F12 medium (without bicarbonate and without red phenol;Sigma) was reconstituted following the manufacturer's directives in purewater. For one liter, 1.2 g of sodium bicarbonate was added and the pHmade to 7.40 with NaOH/HCl. This solution was filtered, distributed in500 mL sterile bottles and stored at 4° C. for a maximal period of threemonths.

[0114] Cell Culture Maintenance Medium:

[0115] Basal DME-F12 medium was supplemented with 10% (v/v) FBS (fetalbovine serum), 100 U penicillin G/50 μg streptomycin sulfate/mL medium,2 mM L-Glutamine (Sigma) and 1 nM E₂.

[0116] Experimental Medium:

[0117] Basal DME-F12 medium was supplemented with 5% FBSA (fetal bovineserum adsorbed on dextran-charcoal), 2 mM L-Glutamine, 100 U penicillinG/50 μg streptomycin sulfate/mL medium and 50 ng/mL insulin (Sigma). Asdescribed for the ZR75-1 cells, solid extract and E₂ were added at thesame concentrations.

[0118] Preparation of FBSA:

[0119] Fetal bovine serum was mixed with 1% (w/v) charcoal (carbondecolorizing alkaline). A solution of dextran T70 was added to thecharcoal-serum solution to achieve a concentration of 0.1% (w/v). Themixture was agitated overnight at 4° C. After centrifugation at 4° C.for 30 minutes at 10,000× g, the serum was decanted, mixed again withthe same proportions of charcoal and dextran, agitated at roomtemperature for three hours and re-centrifuged. The serum was thenheat-inactivated at 56° C. for 20 minutes, sterile filtered andaliquoted in sterile conical Falcon tubes.

[0120] Experimental Culture Assays and Results:

[0121] ZR75-1 and MCF-7 cells were grown to reach a density ofpopulation of 20 000 cells/well on 24-well plaques or 150 000 cells/wellon 6-well plaques, and treated in the presence or absence of differentconcentrations of solid extract as prepared above. To this effect, thesolid extract is resuspended in culture medium and sterile filtered, sothat hydrosoluble components thereof are recovered and tested. Allexperiments have been performed in triplicates. Culture media have beenwithdrawn and replaced by fresh media every two days. Cells were grownin an incubator under a constantly humidified atmosphere containing 5%CO₂, at 37° C., for 17, 7, 3 or 3 days, corresponding to the first,second, third or fourth experiment, respectively. Cell growth inhibitionwas measured by direct counting of the cells or by measuring the totalDNA content of a well. TABLE 1 Cell Inhibition (%) Concentration ofsolid extract MCF-7 ZR75-1 1^(st) experiment: 17 days  1 mg/ml 1.5 2.0 5 mg/ml 14.33 33.6  10 mg/ml 62.66 90.8 2^(nd) experiment: 7 days  1mg/ml 3.73 0.97  5 mg/ml 15.7 29.0  10 mg/ml 68.37 66.0 3^(rd)experiment: 3 days  50 mg/ml 95.8 95.0 100 mg/ml 94.6 98.0 4^(th)experiment: 3 days  10 mg/ml 34.4 51.5  20 mg/ml 62.5 70.5  50 mg/ml95.8 95 100 mg/ml 94.6 98

[0122] The above percentages of inhibition of cell growth demonstratethat the solid extract can inhibit in a dose-dependent manner the growthof these two cell lines.

[0123]FIG. 2 shows that doses of 50 and 100 mg/mL of the solid extractclearly provoke hypoplasia on these cell lines, after three days oftreatment.

[0124]FIG. 3 shows that, in the presence of 10⁻¹² to 10⁻⁹ M E₂, treatedcells respond like control cells by being non-stimulated by thesehormone dosage rates. However, above 1 nM, control cells are stronglystimulated, and concentration of DNA reaches 3.75 μg in the presence of10⁻⁷ M E₂ (versus 0.69 μg in control without E₂). In cells treated with30 and 50 mg/mL of solid extract, DNA measured at the maximalstimulation is 1.9 and 1.8 μg, respectively. FIG. 3 shows that theaffinity constant (Km) of the treated cells for E₂ is 3 and 16 timeshigher (31.3 nM and 174.0 nM) than the value of Km of the control cells(11.7 nM), in the presence of 30 and 50 mg/mL of solid extract,respectively. This means that higher concentrations of E₂ are necessaryto achieve the same growth of the cells when the solid extract ispresent. Therefore, this extract diminishes the maximal response (90%inhibition thereof) and increases the affinity constant of the treatedcells to E₂. The results with the solid extract are indicative of ahydrosoluble component remaining entrapped therein, and which isextractable in an aqueous phase. Hydrosolubility is a key feature thatshould normally be satisfied prior to achieving a biological activity.

[0125] In vivo Assays:

[0126] DMBA Induced Rat Mammary Breast Cancer Model

[0127] Description of the Test System:

[0128] Four hundred 40 day old female Sprague-Dawley rats (purchasedfrom Charles River Co., St-Constant, Quebec, Canada) were adapted totheir environment for 12 days. At that time, 20 mg DMBA in 1 mL corn oil(9, 10-Dimethyl-1,2-Benzanthracene; purchased from Sigma Chemical Co.)was administered by gavage. Three months after this treatment, 240 ratshaving developed a mammary breast cancer have been selected anddistributed in two groups. The first group consisted of five sub-groupsof rats. The rats of the treated groups were given a daily dose ofincreasing concentrations of the solid extract in 3 mL of water foreight weeks while the control group received the same volume of water.The second group consisted of four sub-groups of rats. The rats of thetreated groups were also given a daily dose of the solid in 3 mL ofwater combined with or without the liquid extract, for ten weeks whilethe control group received the same volume of water. Only one sub-groupof the second group of rats treated with a concentration of 3000mg/Kg/day of the solid and 3 mL of the liquid extract was also given anintraperitoneal (i.p.) injection of a smaller dose of the liquid extract(about 8 mg of protein in 1 mL of water).

[0129] Rats weighed 151-175 g at the beginning of the two experimentsand received food and water ad libitum. The first group of rats hadtumors of average diameter of 0.9 cm while the second group of rats hada tumor of average diameter of 0.6 cm.

[0130] b. Anti-Tumor Activity:

[0131] The results are summarized as shown in Table 2: TABLE 2 Dailydoses of solid extract % tumor growth inhibition (decrease ofadministered by gavage tumor diameter versus control) 1^(st) experiment:duration 8 weeks Placebo  0 500 mg/Kg/day  2 1000 mg/Kg/day  4 3000mg/Kg/day 14 5000 mg/Kg/day 15 2^(nd) experiment: duration 10 weeksPlacebo  0 3000 mg/Kg/day 12 3000 mg/Kg/day 3 mL liquid 18 extract 3000mg/Kg/day + 3 mL liquid 20 extract + 1 mL liquid extract i.p.

[0132] These results demonstrate that the solid extract contains anactive component that is absorbed in the gastrointestinal tract andslows down tumor progression. This inhibition might be a direct effecton the tumor cells or an anti-angiogenesis mediated effect interferingwith tumor growth.

[0133] The liquid extract also contains inhibitory activity since itsadministration caused an additional reduction of tumor size of about 6%.

[0134] These results suggest that the solid extract contains residualhydrosoluble active components, which could be re-extracted in anaqueous solution to recover hydrosoluble components maximally, if theyield can be still improved.

[0135] c. Histopathology:

[0136] For evaluating the non-toxicity of the solid extract, the animalsused in the above in vivo experiments were killed by decapitation andthe following tissues were taken for analysis: liver, lung, kidneys,heart, brain, muscle and mammary glands. Fat was taken out of thesetissues. After that, they were fixed for two days in Bouin fluid. Afterdehydration in ethanol, the fixated tissues were embedded in paraffin.Sections thereof were obtained and mounted on glass slides, stained withhaematoxylin and visualized under microscope.

[0137] The histological examination revealed that no deleterious effectwas visible when using the largest doses of solid extract alone or whenusing the solid extract in combination with the liquid extract (data notshown).

[0138] This suggests that the solid extract and the liquid extract havea selective tumor size regressive activity.

[0139] In mammary gland tumors (see FIGS. 4a and b), an importantdiminution (55%) of the area of blood vessels was observed in the groupof rats having received solid and liquid cartilage extracts (FIG. 5).

[0140] The diminution of the tumor size might be due to an importantdecrease in its vascularization, to a direct effect on tumor cells, or acombination of both phenomena. The anti-angiogenic effect of theseextracts is well depicted above. The direct hypoplasiant effect has beenobserved in vitro on hormone-dependent cells, which has been confirmedin vivo.

[0141] Because the above-mentioned results showed that the liquidextract had an increasing effect over and above the effect of the solidcartilage extract on ZR75-1 cells, the components thereof were furtherinvestigated.

[0142] Liquid Extract

[0143] In vitro Assay:

[0144] Tumor Cell Lines:

[0145] Several tumor cell lines were grown in the presence of liquidcartilage extract to examine whether the hypoplasiant activity observedwith the solid extract (above section) was present in the liquidextract.

[0146] Briefly cells were plated in 96 well plates and grown in culturemedia (specific for each cell type; for example, MCF-7 cells were grownas described in above section) in presence or absence of variousconcentrations of liquid extract. Cell proliferation was measured usinga MTT assay following 3 to 5 days of culture. The tumor cell lines were:CHANG Tumor hepatocytes Hep-G2. Tumor hepatocytes A2780 Ovarianadenocarcinoma cells MCF-7 Breast adenocarcinoma cells (estrogendependent) MCF-7-ADR Adriamycin-resistant breast adenocarcinoma cells.

[0147] The liquid cartilage extract showed antiproliferative activity onall tumor cell lines. The strongest inhibitions, 50 and 80%, wereobtained at a concentration of 8.5 mg/mL (dry weight of liquidextract/mL of culture medium) on MCF-7 and A2780 cells, respectively.

[0148] Primary Cultured Cells:

[0149] Fibroblasts from Neovascular Glaucoma:

[0150] In order to evaluate the specificity of biological activity ontumor cells, the liquid extract was tested on mesenchyme originatingcells, human TENON fibroblasts (HTFs), which are normal fibroblasts.HTFs from two patients (one with neovascular glaucoma, NVG, and theother with primary open angle glaucoma, POAG) have been used.

[0151] Subculturing and Maintenance of HTFS:

[0152] Each confluent culture was passaged by washing and detaching with-0.5 mL of 0.05% trypsin/0.5 mM EDTA (Gibco 610-5300 AG) for 5-10minutes at 37° C. DME/F-12 medium (1.5 mL) containing 15% fetal bovineserum (FBS) was then added to neutralize trypsin/EDTA.

[0153] Association of the cells was made by triturating and transferringinto 25 cm² T-flasks, into which additional medium containing 10%(FBS)was added. After confluence was reached, the HTFs were transferred into75 cm² and eventually, into 180 cm² T-flasks. When enough cells wereobtained, some cells were utilized for the experiments as describedbelow, and others were simultaneously frozen to preserve identicalpassages for future experiments.

[0154] Experimental Protocols:

[0155] When confluence was reached, cells from one patient growing intwo or three identical 180 cm² T-flasks were dissociated by theprocedure described above. After a short low speed centrifugation, theywere counted with a ZMI Coulter Counter 216013, equipped with a256-Channelyzer.

[0156] For all the in vitro experiments that follow, approximately fiftythousand cells were inoculated in 1 mL of DME/F-12 medium containing 1%FBS into each 16 mm dish and a 12-well plate. Seventeen hours (hrs)after seeding, 1 mL of fresh identical medium supplemented with 1% FBS(“absolute” controls) was added. Depending on the experimental design(see above and below), the 1% FBS medium was supplemented or not withGFs (Growth Factors) or with the liquid cartilage extract, and sterilefiltered. On this day (day 0), some samples of cells were also countedto determine plating efficiency (which should be equal or greater than95%).

[0157] Forty-eight hours after the onset of the experiments, the cellswere rinsed, dissociated and counted again. The number of cells wasexpressed as a percentage of that obtained in the “absolute” controls.

[0158] Each “absolute” control, containing 1% or 5% FBS, respectively,and each experimental group, supplemented with 1% FBS and with anindividual GF or liquid cartilage extract consisted of triplicatesamples.

[0159] Each experiment was carried out on the cells of one or twopatients at a time, and was repeated at least twice.

[0160] In these experiments, GFs, porcine platelet-derived growth factor(PPDGF) and human recombinant basic fibroblast growth factor (hr bFGF)were added in concentrations of 10 to 100 ng/mL in 1% FBS, respectively.Forty-eight hours after the onset of the experiment, the cells weredispersed by Trypsin-EDTA and counted on the Coulter counter. Alltriplicate values (columns 1, 2 and 3) appearing below equal onetwentieth of counts per well.

[0161] Results:

[0162] The results are summarized in FIG. 6. HTFs were obtained from theglaucoma of a 53 year old man. While growth factors like PDGF and bFGFshowed a stimulating activity on HTFs (* P<0.02, ** P<0.01; determinedby Student-Fisher Test), no effect, positive or negative, was obtainedwhen these cells were grown in the presence of liquid cartilage extract(1 Kg/2L). This suggests that the hypoplasiant activity of the liquidcartilage extract on tumor cells is not universal and does not affectthe growth of fibroblasts. The same cartilage extract neither had aneffect on another type of fibroblast cells, HSF (Human Skin Fibroblasts;data not shown). Even though not tested, it is assumed that the solidextract also produces no effect on normal cells.

[0163] Endothelial Cells from Human Umbilical Vein (HUVECs):

[0164] HUVECs were extracted with collagenase-controlled digestion asdescribed in Jaffe et al. (1973). Pure endothelial cells were usedbefore the fourth passage (trypsin-EDTA at each passage). Quality of thecells were analyzed for their capacity to incorporate di-acetylated LDLand to be labeled with factor VIII.

[0165] Endothelial cells were plated at a density of 2 500 cell/cm² intosterile dishes coated with gelatin. Cells were cultured with completemedium (Medium199+heparin (90 μg/ml)+L-glutamine (2mM)+bicarbonate+Fetal Bovine Serum (10%)+Endothelial Cell GrowthStimulant (120 μg/ml)) during 24 h to insure cell adhesion. Then, thecells were washed 3 times with PBS and the culture medium was addedaccording to experimental conditions. The last PBS wash was consideredas time 0.

[0166] Each experiment was performed in triplicate and statisticanalysis was performed for comparison. Culture medium was changed after24 h, and every other day. After 168 h of culture, BrdU (10 mM final)was added to each culture media and incubated 2h at 37° C. Then, cellswere detached with short trypsin-EDTA digestion and transferred into96-well plates to allow ELISA detection of BrdU. ELISA was performedwith a kit from Boehringer Mannheim. A control was performed withoutcells to determine the background. Another control was performed bymeasuring the DNA content in the culture medium at the end of theincubation period to sort out whether the liquid cartilage extractaffected cellular adhesion.

[0167] Cell proliferation was also evaluated with the amount of DNApresent in the petri dishes. Each experiment was performed in triplicateand statistical analysis was performed for comparison. Culture mediumwas changed daily. After 168 h of culture, cells were lysed withNa-Citrate-sodium dodecyl sulfate solution and incubated with theHoescht 33358 dye. Samples were read at 365 nm with aspectrofluorometer.

[0168] Finally, the amount of cells present in petri dishes wasevaluated by measuring acid phosphatase activity. Each experiment wasperformed in triplicate and statistical analysis was performed forcomparison. The activity of this enzyme showed a strong correlation withthe number of endothelial cells in petri dishes (BrdU incorporation andHoescht labeling; data not shown). Acid phosphatase activity wasmeasured with a kit from Sigma Chemical Company.

[0169] Results:

[0170] The results demonstrated a dose-response inhibition ofendothelial cell proliferation with the liquid cartilage extract (FIG.7). The ED50 obtained is approximately 90 μl of liquid cartilage extract(equivalent to approximately 1.5 mg dry weight present in the liquidextract).

[0171] Keratinocytes:

[0172] Liquid cartilage extract was tested in keratinocytes whichProtein Kinase C (PKC) was activated by triphorbol acetate (TPA), aknown agonist of this cellular transduction pathway.

[0173] Normal human epidermal keratinocytes were established as primarycultures (Matsui et al. (1992) J. Invest. Dermatol. 99: 565-571).Cultures were grown in a serum-free defined medium (KGM) containingepidermal growth factor (10 ng/ml), insulin (5 μg/ml), hydrocortisone(0.5 μg/ml) and bovine pituitary extract (70 μg/ml) in a modified MCDE153 formulation.

[0174] Keratinocytes were grown to 70% confluence, and 48 h afterrefeeding with fresh medium, treated with either 200 ng/mL TPA or 2μl/mL DMSO with no additional refeeding. Various concentrations ofliquid cartilage extract were added or not to the culture medium.

[0175] Results:

[0176] The results showed no effect of the liquid extract onkeratinocyte proliferation; it also had no effect on TPA-inducedinhibition of proliferation. However, liquid cartilage extract was ableto inhibit TPA-induced keratinocyte differentiation (FIG. 8). The levelof differentiation of the keratinocytes was increased 5-fold by TPA.Liquid cartilage extract by itself had no effect on cornified envelopeformation. However, liquid cartilage extract inhibited TPA-inducedcornified envelope formation by more than about 60%.

[0177] Recent publications have shown that PKC activation led normalkeratinocytes to produce increased amounts of interleukin-8 (IL-8), amediator of inflammation (Chabot-Fletcher et al. (1994) J. Invest.Dermatol. 103: 509-515). Moreover, psoriatic keratinocytes produce veryhigh amounts of IL-8, which further promote neovascularization inpsoriatic plaques (Nickoloff et al. (1994) Am. J. Pathol. 144: 820-828).It therefore appears that psoriasis has at least two key etiologies:inflammation and angiogenesis. Other possible etiologies includemetalloprotease activity and endothelial cell proliferation. Othercytokines, growth factors and integrins are also involved in psoriasis.

[0178] It is not known whether TPA-induction mimics psoriatickeratinocytes. If such is the case, these results suggest that cartilagemay have no deleterious effect upon normal keratinocytes in vivo, whileit may have an effect on psoriatic (or activated) keratinocytes.Inhibition of the production of IL-8 in TPA-activated keratinocytes aswell as in psoriatic plaques or keratinocytes by the liquid cartilageextract remains to be verified. Decreased IL-8 levels and/or othergrowth factors might explain the anti-inflammatory and anti-angiogeniceffects of this extract.

[0179] Collagenase Assay:

[0180] This assay is described in Knight et al. ((1992) FEBS Let. 296,263-266). The method utilizes a fluorogenic peptide substrate(Mca-pro-leu-glu-leu-Dpa-ala-arg-NH₂) mimicking the active site ofmetalloproteinases. This substrate has a fluorescent group (Mca) at oneend and a fluorescence quenching group (Dpa) at the other. In the intactsubstrate, the quenching group effectively masks the fluorescence. Uponenzyme cleavage of the substrate, the fluorescence in the test tubeincreases.

[0181] Collagenase activation is described in Weingarten et al. (1985)in Biochemistry vol. 24, p. 6730. 1 μg was diluted to 100 μl with 50 mMTris-HCl, 10 mM CaCl₂. The pH 7.5, 1 μl at 10 mg/mL solution of trypsin(in 1 mM HCl) was added and incubated for 15 min at 20° C. Activationwas terminated by adding 10 μl of soybean trypsin inhibitor (SBTI, 5mg/ml). To each microcuvette was added:

[0182] 25 or 50 μl inhibitor* (made up to 50 μl with water);

[0183] 40 μl 50 mM Tris-HCl, 200 mM NaCl, 10 mM CaCl₂, pH 7.5;

[0184] 8 μl activated collagenase** (67 ng final); and

[0185] 2 μl substrate (1 mM stock solution in DMSO, 20 μM final).

[0186] Fluorescence was recorded at λex=328 μm, λem=393 nm.

[0187] *: the inhibitor is defined as a control substance (such as EDTAor Ortho-phenanthrolene) or the liquid cartilage extract.

[0188] **: the collagenase is defined as human type I, type IV, andamphibian tadpole collagenase; gelatinase has also been used.

[0189] Twenty-one ng of activated collagenase was added to 5 μg of calfskin collagen (Worthington) +/−inhibitor in a final volume of 20 μl.Reactions were incubated for 16 h at 35° C., then stopped by addingSDS-PAGE sample with 40 mM EDTA, boiled and loaded on a 8%polyacrylamide gel.

[0190] Results:

[0191] The results obtained with liquid cartilage extracts showed adose-response inhibition of collagenase activity. FIG. 9 shows resultsobtained with collagenase assay. The ED₅₀ is obtained with 30 μl ofliquid extract (or 0.51 mg dry weight present in 30 μl of liquidextract).

[0192] Assays for Evaluating the Interference with VEGF:

[0193] The formation of new blood vessels is essential for a variety ofphysiological processes. It requires the co-operation of a variety ofmolecules that regulate cellular processes such as extracellular matrix(ECM) remodeling, invasion, migration and proliferation. (Ingber, Sem.Cancer Biol. (1992) 3: 57-63; Schwartz, S. M. et al. (1993) J.Cardiovasc. Pharmacol. 21(Suppl. 1): S31-S49; Auerbach, W. et al. (1999)Pharmac. Ther. 1994; 63:265-311; Moses, M. A. et al. (1995) Int. Rev.Cytol. 161:1-48; Brooks, P. C. (1996) Cancer Metastasis Rev.15:187-194.) Abundant evidence also suggests that angiogenesis ispreceded and/or accompanied by enhanced microvascular permeability.Vascular permeability is also one of the key events of inflammation.This means that collagenolysis, inflammation and angiogenesis sharecommon mechanisms or at least have mechanisms that are inter-related.

[0194] In general terms, angiogenesis is organized into three majorphases: an initiation phase, a proliferative/invasive phase and adifferentiation/maturation phase (Brooks, P. C. (1996) Eur. J. Cancer32A: 2423-9). The initiation phase can be triggered by activation ofvascular cells via a variety of angiogenic cytokines and otherphysiological mediators (Klagsbrun, M. et al. (1991) Annu. Rev. Physiol.53:217-32; Ferrara, N. (1995) Nature 376-467; Koch, A. E., et al. (1995)Nature 376:517-19; Bussolino, F. et al. (1996) Eur. J. Cancer 32A:2401-12; Mazure, N. M. et al. (1996) Cancer Res. 56:3436-40; Ferrara, N.et al. (1997) Endocrine Rev. 18:4-25; Sandner, P. et al. (1997) KidneyInt. 51:448-53). A partial list of the more well-characterized growthfactors known to promote angiogenesis include basic fibroblast growthfactor (bFGF), vascular endothelial growth factor (VEGF),platelet-derived growth factor (PDGF) and tumor necrosis factor alpha(TNF-alpha). These cytokines and other angiogenic molecules can bereleased from a number of sources, including endothelial cells,inflammatory cells, mast cells and macrophages, as well as from avariety of tumor cells (Knighton, D. R. et al. (1983) Science221:1283-85; Scheweigerer, L. et al. (1987) Nature 325:257-9; Hamada, J.et al. (1992) Br. J. Cancer 66:349-354; Rak, J. E. et al. (1994) J. CellPhysiol. 159:245-55; Rak, J. et al. (1996) Eur. J. Cancer 32A: 2438-50;Moses, M. A. (1995) et al. Int. Rev. Cytol. 161:1-48; Proost, P. et al.(1996) Int. J. Clin. Lab. Res. 26:211-23; Polverini, P. J. (1996) Eur.J. Cancer 32A:2430-7; Ferrara, N. et al. (1997) Endocrine Rev. 18:4-25).The proliferative/invasion phase of angiogenesis is characterized bycell replication (Form, D. M., et al. (1986) Lab. Invest. 55:521-530),re-organization of proteins of the cytoskeleton and those involved inmembrane adhesion (Pepper, M. S. et al. (1992) J. Cell Physiol.152;196-205; Klein, S. et al. (1993) Mol. Biol. Cell 4:973-82; Klein, S.et al. (1996) J. Biol. Chem. 271:22583-90) and production of proteasesthat are secreted to promote endothelial cell migration in thesurrounding matrix (Mignatti, P. et al. (1994), J. Cell. Biol.113:1193-201). Finally, the differentiation/maturation phase ischaracterized by endothelial cell production of a basement membrane,lumen formation and junctional coupling with other cells (Pepper, M. S.et al., Biochem. Biophys. Res. Commun. (1992) 189:824-31; Bischoff, J.(1995) Trend Cell Biol. 5:69-74).

[0195] VEGF is a multifunctional angiogenic growth factor. It stimulatesthe endothelial cells to proliferate and to migrate. It is also apermeabilising agent that renders the endothelial tissue hyperpermeable,leading to the extravasation of plasma proteins. This conducts to atransformation of the extracellular matrix and a stimulation ofangiogenesis.

[0196] Accordingly, the effect of the present shark cartilage extract onthe VEGF activity was established both in vivo and in vitro: it blocksthe stimulation of endothelial cell proliferation, it competes with thebinding the VEGF to its receptor, and it blocks the extravasationinduced by VEGF. Therefore the shark cartilage extract (also calledAE-941) has anti-metalloprotease, anti-angiogenic and anti-inflammatoryactivities that appear to be due in part to its action on VEGF activity.Endothelial cell proliferation

[0197] Human Umbilical Vein Endothelial Cells

[0198] (Clonetics Corp.; San Diego, Calif.) were cultured inM199-bicarbonate buffered medium supplemented with heparin 100 μg/ml,L-glutamine 2 mM, ECGS 20 μg/ml, 10% FBS, and penicillin-streptomycin(1%). Four thousand cells were seeded in 96-well sterile culture dishesand cultured for a 6- to 8-hour period to allow cell attachment.Afterward, the cells were starved for 24 h in M199-bicarbonate bufferedmedium supplemented with 2% FBS and antibiotics. Then, fresh medium (2%FBS and antibiotics) containing increasing concentrations of VEGF orbFGF was added to the cell cultures. Cells were incubated for 3-dayperiod. Cell number was then evaluated by a colorimetric assay using thecell proliferation reagent tetrazolium salt WST-1 according to theprocedure established by the manufacturer (Boehringer Mannheim).

[0199] Bovine Artery Endothelial Cells (BAEC):

[0200] The BAEC were isolated from fresh aortas in our laboratory. Theiridentification was established by their cobblestone monolayermorphology, factor VIII immunochemistry and by negative smooth musclecell α-actin staining. The BAEC were cultured in DMEM supplemented with5% FBS and penicillin-streptomycin (1%).

[0201] Results:

[0202] The presence of functional VEGF receptors expressed in HUVEC isshown by a dose-dependent stimulation of the proliferation by VEGF (FIG.28a). Moreover, it was demonstrated that the present shark cartilageextract can inhibit the proliferation of HUVEC induced by VEGF (10 ng/mL) (FIG. 28b). The resulting IC₅₀ of the cartilage extract

-941 in VEGF-stimulated HUVEC corresponds to 2.55 mg/mL. TABLE 3 Kd andBmax of VEGF and bFGF receptors in HUVEC Kd Bmax Binding site (nM)(fmol/10 cells) VEGF 0.12 2.6 bFGF 0.36 4.5

[0203] Radioreceptor Binding Assay with ¹²⁵I-VEGF or ¹²⁵I-bFGF

[0204] Binding studies were performed on HUVEC and BAEC grown atconfluence in 24- or 12-well tissue culture plates, respectively. Thebinding buffer was M199 media supplemented with 5 mM Hepes and 0.1%gelatin at pH 7.3. Saturation of VEGF and bFGF receptors were determinedby adding 0.2 mL (for HUVEC) or 0.5 mL (for BAEC) of binding buffercontaining increasing concentrations of ¹²⁵I-VEGF or ¹²⁵I-BFGF (0.05-10mg/well) to the endothelial cells in culture for a 2-hour period at 4°C. Non-specific binding was determined at each concentration by adding a100-fold excess of unlabeled VEGF or bFGF. At the end of incubation,cells were washed twice with ice-cold M199 containing 1 mg/mL of bovineserum albumin. The cells were collected after trypsin incubation, andthe radioactivity evaluated on a y counter. Binding site competitionstudies for ¹²⁵I-VEGF or ¹²⁵I-bFGF (4 ng/well; a concentration close tothe saturation) was evaluated in the presence of increasingconcentrations of unlabeled VEGF or bFGF (0-400 ng/well), or cartilageextract (0-7.2 mg/well). Each determination was performed in triplicate.

[0205] Results:

[0206] Competitive binding assays of the present cartilage extract forVEGF were conducted in HUVEC and bovine artery endothelial cells (BAEC).Results indicated that present cartilage extract competes with thebinding of VEGF to its receptor while it did not for bFGF (FIGS. 29a and29 b), although it inhibits bFGF mitogenic activity (not shown). TABLE 4Competition of

-941 to the binding of VEGF and bFGF to their respective receptorbinding sites Radioligand Cold ligand IC₅₀ ¹²⁵I VEGF (4 ng) VEGF 0.48 nM¹²⁵I-bFGF

-941 2.71 mg/mL ¹²⁵VFGF (2 ng) bFGF 0.57 mM ¹²⁵I-bFGF

-941 Not applicable

[0207] Inhibition of VEGF Induced Extravasation

[0208] This study aimed at determining whether oral administration of

-941 could inhibit the extravasation of Blue Evans (BE) dye in mice.Toward this end, mice were orally treated with

-941 (150 mg/ml) or saline prior to I.V. administration of VEGF (0.1nmol/kg) and BE (20 mg/kg).

[0209] Results:

[0210] The amount of BE present in various organs 10 min after VEGFadministration was illustrated in FIG. 30. These results indicate thatoral administration of

-941 cartilage extract in mice significantly reduces the induction ofextravasation by VEGF and support its oral bioavailability.

[0211] Inhibition of VEGF Receptor Phosphorylation

[0212] The effect of the liquid extract upon VEGF receptorphosphorylation was determined to elucidate a possible mechanism bywhich the cartilage extract blocks the action of VEGF. The effect ofVEGF on VEGF-receptor phosphorylation was determined on BAEC that werepre-treated with the liquid extract.

[0213] BAEC were treated during 18 hours in a serum free medium in thepresence or absence of the cartilage extract (7 mg/ml; 50%). Cells werethen stimulated with VEGF (1 nM) for one minute or CNF₁ toxin (1 μg/ml)for six hours. Afterward, the cells were lysed and the VEGF receptorswere immunoprecipitated with antibodies specific for either the type 1or type 2 receptors. The degree of phosphorylation of these tworeceptors was determined by immunoblot with an anti-phosphotyrosineantibody.

[0214] Results:

[0215] As illustrated in FIG. 31, the liquid extract reduced the abilityof VEGF to stimulate VEGF-receptor phosphorylation by approximately 50%.This effect appears more pronounced for the type 2 VEGF-receptor thanfor the type 1 VEGF-receptor. Similar results were obtained with theCNF₁ toxin, which induces the phosphorylation of type 2 VEGF-receptor.These results indicate that the liquid extract contains components thatinteract directly with the VEGF-receptor.

[0216] Other Anti-Metalloprotease Activities:

[0217] Other molecules that are targeted by the components of thepresent cartilage extract have been investigated. The anti-angiogenicand anti-collagenolytic activities of the latter indicates that ananti-matrix metalloprotease activity is present therein. Other proteasesincluding other matrix metalloproteases have been tested for theirputative role as targets for the cartilage extract. By fluorometricassays, strong inhibition has been observed against gelatinolytic andelastinolytic activities for gelatinase A (MMP-2), gelatin B (MMP-9) andagainst the elastinolytic activity of metalloelastase (MMP-12), porcinepancreatic elastase (PPE) and human leucocyte elastase (HLE). Zymographyalso revealed a strong inhibition of gelatinolytic activity of MMP-2 andMMP-9 and of caseinolytic activity of MMP-12. No inhibition wasmeasurable against urokinase and plasmin proteolysis, which indicates aspecificity of the inhibitory activity against matrix-degradingproteases. The gelatinolytic activities of a variety of molecules areinhibited by the cartilage extract (amongst them, MMP-7, MMP-13,papain). The cartilage extract comprises an inhibitor capable of bindingantibodies directed against human tissue inhibitors of metalloproteases(TIMPs). These results suggest that the cartilage extract comprisesTIMP-like inhibitors. A TIMP crossreactive molecule of molecular weightof about 31 KDa has been identified in the liquid cartilage extract byWestern blotting (see FIG. 32). The 31 KDa entity strongly reacts withanti-TIMP-2 antibody, but also with anti-TIMP-1 and -3 antibodies.Serine elastases like PPE and HLE are inhibited by the cartilageextract, while TIMPs are ineffective against serine elastases.Therefore, a TIMP-like inhibitor as well as a serine elastase inhibitorappear to be present in the cartilage extract. The cartilage extract isa more potent inhibitor of the elastinolytic activity than of thegelatinolytic activity of elastase, MMP-2 and MMP-9, which suggests thatit comprises an analogue of elastin.

[0218] Ex vivo Assays:

[0219] Embryonic Vascularization Test (EVT):

[0220] Definition of the Best-System:

[0221] The normal development of the chick embryo involves the formationof an external vascular system located in the vitelline membrane thatcarries nutrients from the vitellus (yolk of an egg) to the developingembryo. When placed onto the vitelline membrane, anti-angiogenicsubstances can inhibit the blood vessel development that occurs in thevitelline membrane. To facilitate access to the vitelline membrane,chick embryos are transferred to a sterile culture box (Petri dish) andplaced in a humidity- and temperature-controlled incubator. Embryos canthen develop in this ex ovo condition for several days.

[0222] An aliquot of liquid cartilage extract is mixed with amethylcellulose solution and allowed to air-dry into thin discs. Duringthis procedure, intrinsic NaCl present in the liquid cartilage extractinterferes with the EVT when the amount of liquid cartilage per disc isover 25 μg. Therefore, desalinating the liquid extract may be necessary.Dialysis with a membrane cut-off smaller to 100 Daltons orelectrodialysis have been found acceptable methods.

[0223] Methylcellulose forms an inert matrix through which the liquidextract can diffuse slowly. Methylcellulose discs containing the liquidextract are placed on the external border of the vascular perimeter ofthe vitelline membrane where the angiogenic process is still active.

[0224] The effect of discs-containing liquid cartilage extract onproximal vascular development is always compared to that ofdiscs-containing water plus equimolar amount of NaCl. The discs areplaced on the embryo's vitelline membrane on Day 0 or Day 1 of the exovo growth process; at this point, only beginnings of the main bloodvessels are invading the vitellus. The embryos are then put in cultureconditions until vascularization is assessed (approximately 24 h).Water- and liquid extract- containing discs are always simultaneouslyadded on the vitelline membrane of the same embryo. Both discs arearranged in a symmetric fashion with respect to the cephalo-caudal axisof the embryo in order to minimize inter-individual variations whencomparing shark cartilage extracts with controls.

[0225] Anti-Angiogenic Activity:

[0226] EVTs were performed using different concentrations of protamine(37, 75 and 150 μg) as a positive control or liquid cartilage extract.After one day of culture, the level of vascularization in the areacovered by the disc is graded by a pair of scientists in the usual blindfashion. To facilitate the location of the discs, a black O-ring isplaced around it just after its deposition on the vitelline membrane.Evaluation scale for the EVT-test is based on the 1-2-3 score (score=3).Normal vascularization when compared to the opposite horizontal quadrantor the matching quadrant of a control embryo (score=2). Blood vesselsenter the area covered by the disc, but vanish at mid-course. Majorblood vessels cross the area covered by the disc but their trajectory isclearly affected or a decrease in the lateral branching density isobserved (score=1). No blood vessels are observed in the area covered bythe disc, or their path is rapidly deviated to avoid the area covered bythe disc. Blood vessels do not grow beyond the area covered by the discexcept if they bypass the latter and go beyond it.

[0227] A dose-response inhibition was obtained with protamine (data notshown) and the liquid cartilage extract (FIG. 10). The ED50 was obtainedwith about 170 μg of dry liquid extract (dry weight present in theliquid extract) Wilcoxon-signed rank statistical test was used tocompare the significance of the differences between the two discs (waterand liquid cartilage extract) placed on the same egg.

[0228] Mouse Mammary Adenocarcinoma Model:

[0229] Description of the Test-System:

[0230] The anti-tumor potential of the liquid cartilage extract wastested with a mouse mammary adenocarcinoma model (allograft). Thetest-system consisted to subcutaneously inoculate BALB/C mice with 1×10⁶DA3 cells. These cells originate from a murine mammary adenocarcinomainduced by 7,12-dimethylbenzanthracene (DMBA). The model was establishedby Daniel Medina (1969) (J. Natl. Cancer Inst. 42: 303-310; ibid. (1976)57: 1185-1189). Inoculated cells slowly grow in vivo and form a solidtumor with a low metastatic prognosis.

[0231] DA3 cells were maintained in RPMI 1640 medium supplemented with 1mM mercaptoethanol, 1 mM Hepes buffer solution, 100 mM Na pyruvate, 200mM L-glutamine, 10 mM nonessential amino acids, 1 M vitamins, 10% fetalbovine serum, 1% penicillin-streptomycin at 37° C. with 5% CO₂. Fortumor induction, cells were grown to 70% confluence in complete mediumand then collected using trypsin-EDTA solution. Cells were thencentrifuged and washed three times with phosphate buffer solution, andresuspended at a dilution of 1×10⁶ cells/0.1 ml.

[0232] DA3-cells inoculated mice (n=15) received daily oraladministration of a shark cartilage liquid extract or a placebo (salinesolution). The treatments began 7 days after DA3 cells inoculation.Various concentrations of liquid extract were tested. The amount ofliquid extract administered is expressed with the amount of dry weightpresent in the liquid extract. The test articles were prepared asdescribed here: liquid extract was lyophilized and resuspended in waterat various concentrations (0.2, 1.5, 3, 10, and 20 mg per 200 μl). Thefinal doses that were administered daily were 10, 75, 150, 500, and 1000mg/kg of body weight.

[0233] Anti-Tumor Activity:

[0234] Results show that the maximum inhibition of tumor progression wasobtained with the oral administration of about 75 mg/kg of liquidextract (FIG. 11). Interestingly, larger doses were less potent. Thissuggests that the liquid extract contains substances that can inhibittumor progression and other substances that can inhibit the action ofthe tumor inhibitors. This phenomenon has already been reported forbiological drugs.

[0235] Finally, intraperitoneal administration of (1 mg/kg) liquidextract shows the same anti-tumor activity as an oral dose of about 75mg/Kg.

[0236] Toxicity:

[0237] With all treatments there was no loss in body weight or liquidextract-related death. There were no symptoms or behaviour changesobserved with daily examination of mice during the treatment period. Atthe end of the treatment, mice were sacrificed and the gross morphologyof all organs was analyzed by a certified pathologist; no abnormalitywas detected. Blood analyses did not show any sign of abnormality.

[0238] Histopathology:

[0239] Tumor histopathology did not reveal any gross changes betweentumor from placebo- or liquid extract-treated mice. The extent of tumorviability was quite high in all groups. Analysis of various organs(lung, liver, kidney, pancreas, stomach, intestine, ovary, breast,brain, and heart) did not reveal any specific alteration that can berelated to the liquid extract.

[0240] Mouse Hypersensitivity Model (CHS):

[0241] Description of the Test System:

[0242] Dinitrofluorobenzene (DNFB) is a powerful skin irritant that caninduce a strong inflammatory reaction in BALB/C mice. The liquidcartilage extract was tested to examine whether it could reduce theinflammation response to DNFB in mouse.

[0243] At day 0, 10 mice were sensitized by painting their belly with 25μl 0.5% DNFB. Mice were challenged on the right ear by painting 10 μl0.2% of DNFB 5 days after sensitization. Ear swelling was measured overseveral postexposure times as an index of tissue irritation.

[0244] Results:

[0245] Oral administration of shark cartilage extract (0-500 KDa 0.2 mLper day) during 5 to 8 days prior to sensitization reduced by 37-64% theinflammatory response. The extract administered after exposure toirritant, e.g., during the elicitation or challenge phase, did not haveany significant effect on the inflammatory response. We have furtherdemonstrated that the liquid extract decreased the level ofpro-inflammatory cytokine in mRNA such as IFN-γ and TNF-α in lymphaticnodes as well as IL-1α in skin of mouse DNFB hypersensitivity model (seebelow) following the p.o. administration of about 175 mg/kg/day of theliquid extract. Interestingly, an increased expression ofanti-inflammatory cytokines such as IL-10 was observed in skin as wellas in lymph nodes. These results, along with the results obtained inacne and psoriasis cases, confirm that the inflammatory response isobserved with per os as well as with topical compositions comprising ashark cartilage extract. The anti-inflammatory activity therefore showsan oral bioavailability.

[0246] Preparation of Liquid Fractions Containing Active Molecules

[0247] In vitro Assays:

[0248] Tumor Cell Lines:

[0249] Preparation of Test-System:

[0250] Shark cartilage was harvested and processed the same as describedabove. After centrifugation, the pellet was discarded and thesupernatant was ultrafiltrated as described, up to the sterilefiltration on 0.22 p-m filter. The so obtained liquid extract wasfurther fractionated by different methods. Tumor cell lines were grownas described in above section.

[0251] FPLC Conditions:

[0252] Column Hiload 26 mm×60 cm Sephacryl S-300. FPLC system: fromPharmacia. All samples were filtered on 0.22 μm filter before loading onthe column. The elution buffer was phosphate buffer saline (PBS)filtered and degazed during 15 minutes. The volume of the loaded samplewas usually 3.2 mL (could be up to 13 ml). The flow rate was 1ml/minute. Fractions of 10 mL were collected. The eluted compounds weredetected by their U.V. absorbance (280 nm). A calibration chart wasobtained by using the MW-GF-1000 calibration kit from Sigma, thiscalibration sample having the same volume as the loaded sample toanalyze (3.2 ml). The elution volume of a sample was deduced from theplotting of the molecular weight of the compounds of the calibration kitversus their elution volume to which was subtracted the void volume ofthe column. The void volume was obtained by injecting dextran blue(MW=2,000,000).

[0253] The fractions were tested on ZR75-1 cells for their activity. Thefractions of interest were identified and their characteristics werecorroborated by further study, described below.

[0254] Additional characterization of the active components of thepermeate was conducted on Rotofor (BioRad 170-2950; seeisoelectrofocalization below) and on Amicon filters of different cut-offvalues to obtain fractions of molecular weight between 10-30 kDa, 30-100kDa and over 100 kDa.

[0255] Isoelectrofocalization Conditions:

[0256] A preparation of shark cartilage liquid extract (46 mL of 1 Kg/L)was dialysed overnight against 4 liters of pure water containing 5%glycerin at 4° C. using a membrane Spectra pore #7 MWCO 3500 kDa(Spectrum 132110). The dialyzed solution was mixed with 2.75 mL ofampholytes (Pharmacia #80-1125-87) pH 3.5-10.0 and 0.5 g CHAPS (SigmaC3023; 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane-sulfonate). Thevolume was completed to 55 mL with pure water. The solution was loadedon Rotofor and isoelectrofocalization was conducted at 4° C., at aconstant power of 12 watts (3000 xi power supply BioRad 165-0554), underconstant water circulation for insuring maintenance of the temperature.At the beginning of the separation, the voltage was 380 volts and theamperage 31 mA. When the amperage was stabilized (at 14 mA), the voltageread 870 volts. The isoelectrofocalization was stopped and 20 fractionswere collected. TABLE 5 FRACTION VOLUME (ml) pH  1 3.7 3.56  2 2.1 4.01 3 2.2 4.18  4 2.3 4.31  5 2.2 4.63  6 2.1 5.03  7 2.5 5.30  8 2.1 5.5  9 2.4 5.81 10 2.5 6.26 11 2.3 7.00 12 2.4 7.29 13 2.4 7.64 14 2.5 7.9415 2.3 8.32 16 2.5 8.62 17 2.4 8.94 18 2.9 9.30 19 3.1 9.88 20 3.610.71 

[0257] The identification of these proteins was made by estimating theirmolecular weight on an electrophoresis gel (Laemmli, U.K. (1970) Nature(Lond.) 227: 680).

[0258] These fractions were four-fold diluted with a loading buffer (seeLaemmli) and 8 μL aliquots were submitted to electrophoresis innon-reducing conditions. FIG. 13 shows the electrophoretic profile ofeach fraction and of the material before isoelectro focalization.

[0259] All the fractions were sterile-bottled under a laminar flow hoodby passing them through a sterile Millipack-60 filter having a porosityof 0.22 μm.

[0260] d. Inhibitory Activity on Tumor Cells:

[0261] Liquid cartilage extract were tested on ZR75-1 cells at differentconcentrations in culture medium. The protein content of the fractionswas evaluated by the Lowry dosage method. The results are summarized asfollows (Tables 6, 7, and 8):

[0262] 1st Test:

[0263] Tests performed on Rotofor fractions (the liquid cartilageextract was concentrated by evaporation).

[0264] Protein Identification: TABLE 6 Fractions Identified IsoelectricPoint Median Value Molecular Weight 7-8-9-10 5.30 to 6.26 5.78 29 +/− 1kDa 7-8-9 5.30 to 6.26 5.68 60 +/− 1 kDa 12-13-14 7.29 to 7.94 7.62 48+/− 1 kDa 13-14 7.64 to 7.94 7.79 35 +/− 1 kDa

[0265] TABLE 7 Fractions Molecular Weight 6 and 7 0.1-2.5 kDa

[0266] TABLE 8 Concentration Inhibition of ZR75-1 Cell tested MolecularWeight Cultures 100 μg/ml MW > 100 kDa  64% 100 μg/ml 30 kDa < MW < 100kDa 114% 100 μg/ml 10 kDa < MW < 30 kDa 127% 400 μg/ml MW < 10 kDa 149%

[0267] FPLC fractions 6 and 7 contain active components of a very smallmolecular weight: 0.1 to 2.5 kDa.

[0268] The hypoplasiant effect of the fractions can be up to 33 000times higher than the one observed with the solid extract.

[0269] e. Further Identification of the Active Components of the Eluate:

[0270] The active fractions (tested on ZR75-1 cells) are retrieved inthe following range of molecular weight, determined by another type ofpurification starting with the same liquid extract (1 kg/L) on a 10 mmdiameter×30 cm length Superose-12 column using the FPLC and rotoforprocedures described above. A flow rate of 1 ml/min was selected.Forty-five 45 fractions of 1 mL were collected. TABLE 9 Fractions 20-21activity in fractions corresponding to a molecular weight of 70 to 120kDa Fraction 22 activity in fractions corresponding to a molecularweight of 60 to 70 kDa Fraction 29-32 activity in fractionscorresponding to a molecular weight of 35 to 46 kDa Fraction 34-35activity in fractions corresponding to a molecular weight of 29 kDaFraction 38-39 activity in fractions corresponding to a molecular weightof 0 to 2.5 kDa

[0271] Collagenase Assay:

[0272] HPLC Chromatography:

[0273] A 980 mL sample of liquid extract (DUP) was filtered through a 10kDa cutoff membrane in a tangential flow ultrafiltration unit (PELLICON,Millipore). The unit was first rinsed with 1 L of water. Final yieldswere 480 mL of >10 kDa fraction and 1.8 L of <10 kDa fraction. The <10kDa was concentrated by cold-finger evaporation to 180 mL (<10-10×).Eight times 100 μl aliquots of <10-10× were loaded onto CDC-S Hexyl, 5μm HPLC column (25×0.94 cm) and eluted first with 100% H₂O at 4 ml/min;then at 8.5 ml/min with 100% MeOH. Fractions were collectedcorresponding to OD₂₁₄ peaks.

[0274] Five fractions were collected (FIG. 14): Fr1, Fr2, Fr3, Fr4, andFr5. The first three fractions include at least a major peak.

[0275] Anti-Collagenolytic Activity:

[0276] Two collagenase assays have been performed to verify whichfraction was the most active to inhibit collagenase. The first assay isthe one performed above, which makes use of a fluorogenic peptidesubstrate (Knight et al. op. cit.). The second collagenase assay isdescribed in Welgus et al. (1979) J. Biol. Chem. 256: 9511-9516. Themethod uses SDS-PAGE to examine cleavage by collagenase type 1 (MMP1).Collagenase type 1 makes a single cut in the native collagen moleculegiving two fragments of 75% and 25% the size of the original collagen.After cleavage for several hours, the reaction is monitored byseparating the products from the substrate by SDS-PAGE. The ratio ofcleaved to uncleaved collagen is assessed visually after staining thegels with Comassie blue (or silver stain).

[0277] Results:

[0278] The results show that Fr1 is the most active fraction to inhibitthe collagenase; a lower level of activity is present in all otherfractions. These results are confirmed notwithstanding the assay thathas been used (fluorogenic assay, see FIG. 14; second assay, see Table10). TABLE 10 COLLAGEN COLLAGEN SAMPLE STAINING FRAGMENT STAININGCollagen only (C) ++++ − C + Enz + +++ C + Enz + EDTA ++++ − C + Enz +DUP + ++ C + Enz + Fr1 ++++ − C + Enz + Fr2 +++ + C + Enz + Fr3 +++ +C + Enz + Fr4 +++ + C + Enz + Fr5 +++ + C + Enz + >10 kDa + +++

[0279] EDTA 40 mM inhibited collagenase. The total liquid extract DUPshowed a low anti-collagenolytic activity. Fractions 1 to 5 were active;the most active was fraction 1. The fraction of a molecular weighthigher than 10 kDa showed no significant inhibitory activity. Therefore,an anti-collagenolytic activity is assigned to molecules having lessthan 10 KDa.

[0280] As mentioned above, the present cartilage extract comprises atleast two types of inhibitors, one type against matrix metalloproteasesMMP-2, -9 and -12, and another type against serine elastases. Thus,although no anti-collagenase activity has been retrieved in thefraction >10 KDa, other inhibitors appear to be present in this highmolecular weight fraction.

[0281] Ex vivo Assay:

[0282] Embryonic Vascularization Test (EVT):

[0283] The liquid cartilage extract was fractionated using a tangentialflow filtration apparatus and a 10 kDa filter (Pellicon, Millipore). Thelower and higher than 10 kDa fractions of the liquid cartilage extractwere tested in the same conditions. They were shown equally potent (FIG.15) in inhibiting neovascularization. This contrasts with theanti-collagenolytic activity which does not appear to be present in thefraction over 10 kDa. Thus the anti-angiogenic activity is due at leastin part to molecules different from an anti-collagenase, e.g., othermetalloproteases or other unrelated molecules.

[0284] Fraction<10 kDa:

[0285] The anti-angiogenic factor in this fraction behaved as theanti-collagenolytic factor during the purification steps describedabove.

[0286] Fraction>10 kDa:

[0287] The fraction was chromatographed on a gel permeationchromatography column (Sephacryl S-300, Pharmacia). A fraction (S300-4)having anti-angiogenic activity was characterized on SDS-PAGE. Theactive fraction (S300-4) had several protein bands having molecularweights of between approximately 8 and 18 kDa (compared with BioRadSDS-PAGE marker proteins). This fraction was further fractionated usinganion exchange chromatography (Mono-Q, Pharmacia) using 25 mm Tris-HClpH 8.0 and a 0 to 1.0 M NaCl gradient. A fraction eluting at between0.8-1.0 M NaCl had high anti-angiogenic activity. Fractions elutingbetween 0.3-0.6 M NaCl and 0.08-0.2 M NaCl had lesser anti-angiogenicactivity.

[0288] Since we are not the first to find a great interest in cartilageextracts, we have verified the unique character of the shark cartilageliquid extract prepared by the present process in side-by-sidecomparison tests with two products described or deducible from the priorart, namely products prepared by the process of Balassa (U.S. Pat. No.4,822,607) and Oikawa et al. (op. cit.).

[0289] Oikawa et al. describe a method by which two main fractions areobtained, one having molecules of molecular weights comprised between 1and 10 kDa, the second having components heavier than 10 kDa. Theyassign anti-angiogenic properties only to the first fraction, the otherbeing said devoid of any anti-angiogenic activity in CAM test. Foradequate comparison of Oikawa's products, we have fractionated our totalliquid extract in two corresponding fractions, and we retained the onehaving 0 to 10 kDa.

[0290] Since Balassa describes a process for extracting a total liquidextract, we have compared our liquid cartilage extract (0 to 500 kDa) tothe product prepared by reproducing Balassa's method, replacing thecalve cartilage by shark cartilage as the starting material.

[0291] We assume that if Balassa and Oikawa describe a processequivalent to ours, the patterns obtained on FPLC, HPLC, and CZE shouldoverlap substantially, and the same anti-angiogenic activity should berevealed on EVT. All samples were made to a final concentration of 12μg/μL (dry weight/volume solution) prior to FPLC and HPLCchromatography. Oikawa's product was centrifuged and filtered prior tochromatography because it contained insoluble material.

[0292] Samples Preparation

[0293] Shark cartilage samples extracted by the three methods werelabelled (with estimated dry weight per volume of solution) as follows:

[0294] 1) DUP is the preparation of the present invention fractionatedto contain molecules between 0 to 500 kDa (12 μg/μL);

[0295] 2) BAL is the preparation according to the recipe of Balassa etal. (12 μg/μL);

[0296] 3) OIK is the preparation of fraction 3 according to Oikawa etal. All samples were made to a final concentration of 12 μg/μL (dryweight/volume) prior to any analysis. The OIK sample had a high amountof insoluble material that could be pelleted readily by centrifuging at13,200 RPM or filtering through a 0.2 μm membrane. Removal by filtrationof insoluble material was essential prior to FPLC, HPLC, and CZE (FIGS.16, 17, 18).

[0297] FPLC Comparison

[0298] Conditions:

[0299] Samples were run on a Superose 12 (10/30) gel permeation columnwith phosphate buffered saline (PBS) as elutent at a flow rate of 0.5ml/min (chart speed=0.25 cm/min). A 100 μl aliquot of the concentrationadjusted samples was filtered through a 0.2 μm membrane beforeinjection. OD₂₈₀ was monitored.

[0300] The column was calibrated with the following standards (MW inDaltons): catalase (232,000), aldolase (158,000), albumin (56,000),ovalbumin (44,000), chymotrypsin (25,700), ribonuclease (13,700),insulin (5,700), insulin B chain (3,500), insulin A chain (2,500)bacitracin (1,450), vitamin B-12 (1,355). Molecular weights of the majorpeaks were calculated by the following equation: log₁₀ MW=7.52-0.212×RT,where RT=elution volume in mL. R²=0.976. Total column volume (Vt) was21.93 mL as determined using cytidine (246 Daltons). Void volume (VO)was determined to be 8.38 mL with blue dextran (2×10⁶ Daltons).

[0301] Results Summary:

[0302] In FIG. 16a, our sample (DUP) had a first major peak (1) whicheluted at 18.76 mL giving a molecular weight of about 3,500 Daltons.Subsequent peaks at 22.7 (2) and 27.3 mL (3) were beyond the totalcolumn volume (21.93 ml, as determined by cytidine). These peaks appearto have some affinity for the column matrix.

[0303] In FIG. 16b), Balassa's sample (BAL) had a small peak (1) elutingnear the Vo of the column (8.4 ml), a peak (2) at 18.5 mL (4,000Daltons) and two peaks eluting after the Vt(3) 22.6 min and (4) 28.2 ml.

[0304] In FIG. 16c), Oikawa's sample (OIK) also had a small peak (1) atthe Vo, peak (2) at 18.9 mL (3,300 Daltons), peak (3) at 21.5 mL (1,000Daltons) and small peak (4) at 27.3 ml.

[0305] In comparing the samples, it is notable that aside from the 3,500Daltons peak, that the major bands of the DUP sample were not observedat the same intensity in the other samples. The OIK sample did appear tohave a small amount of the 27.3 mL peak. The BAL sample had a peakmigrating at 28.2 mL which could correlate with one of the minor peaksin the DUP sample.

[0306] HPLC Comparison

[0307] Conditions:

[0308] C-S-S-hexyl column 5 μm, 25×0.94 cm, CSC #059-085; reverse phasecolumn.

[0309] Results Summary:

[0310] For HPLC on a hexyl-reverse phase column, OD₂₁₀ and OD₂₈₀ weremonitored simultaneously. Fifty μl aliquots of centrifuged samples (allat 12 μg/μl) were loaded and eluted with 100% H₂O. Peaks for eachchromatogram labelled according to OD₂₁₀ (e.g., 1) and correspondingOD₂₈₀ peaks are noted by (e.g., 1). The V₀ of this column was 5.5 mL(1.4 min).

[0311] In FIG. 17a), DUP had 3 major peaks that were observed via OD₂₁₀(1,2,3) and 2 minor peaks (4,5). Two side peaks were observed off ofpeak 1, labelled la and lb. Significant OD₂₈₀ absorbances wereassociated peaks 1, 1a, 1b and 3. In comparison, the corresponding OD₂₈₀absorption for peak 2 is much smaller relative to the OD₂₁₀.

[0312] In FIG. 17b) BAL showed more OD₂₁₀ peaks, but the intensitieswere lower relative to the DUP peaks. As far as overlap of peaks couldgive an indication of identity of molecules, only peaks 3 and 7 in theBAL appear to correlate with the retention times of peaks in the DUPsample (peak 1a or 1b and peak 4, respectively).

[0313] In FIG. 17c), only three major peaks were observed (1,2,3) in OIKextract. Peaks 1 and 3 could correlate to peaks 1 and 3 of DUP samplebut no side peaks of 1 were observed in the OIK chromatogram. The heightof the peaks in the OIK sample were lower than the DUP. Therefore, FPLCand HPLC patterns are characteristic of distinguished products.

[0314] CZE Comparison

[0315] Conditions:

[0316] Apparatus.

[0317] Beckman system (p/ace system 2050) with goal software (version7.11 U); Capillary: Silice (TSPO 50375), 50 μm×97 cm; buffer: 2 M formicacid; Coated solution, 5% p/v hexadimethrene bromide and 2% v/v ethyleneglycol in water; Detector: UV (200 nm); Current: −30 kV; Injection: 0.5psi, 20 seconds; Temperature: 22° C.

[0318] The capillary was conditioned with 1 M NaOH (20 psi, 20 min),water (20 psi, 10 min), Coated solution (20 psi, 20 min), and buffer (20psi, 10 min). Then conditions were settled for a run: Buffer (20 psi, 2min), sample injection (0.5 psi, 20 sec.), run (-30 kV, 45 min), 1 MNAOH (20 psi, 3.5 min), water (20 psi, 3.5 min.), coated solution (20psi, 4 min), and buffer (20 psi, 4 min.).

[0319] Each samples (BAL, DUP, OIK fraction 3) were resuspended at 16.5mg/ml. The pH of each solution was 7.1, 6.8, and 8.2 in BAL, DUP andOIK, respectively. NaCl concentration of each solution was 2.08, 4.37,and 0.71 mg/mL in BAL, DUP, and OIK, respectively.

[0320] Results Summary:

[0321] The molecular profile of each sample (BAL, DUP, and OIK-3) isshown in FIG. 18. The comparison of DUP and BAL samples showed that theBAL sample was contained a larger proportion of peaks with a % area<1.BAL and DUP share the peaks at MT/EOF=1.06, 1.54, 1.59, 1.66, and 3.22.The peaks with the ratio of 1.06, 1.54, and 3.22 have a similar % areain BAL and DUP whereas peaks at ratio 1.59 is 8 times more intense thanin BAL and the opposite is seen at the ratio 1.66.

[0322] DUP and OIK samples present a very different electrochromatogram.OIK has one major peak with several minor peaks. None of these peaks canbe related to one of the DUP sample.

[0323] EVT Comparison

[0324] The anti-angiogenic potential of the samples DUP, BAL and OIK wasanalyzed on EVT (FIG. 19). No significant anti-angiogenic activity wasretrieved in Balassa's extract. The DUP crude extract was compared tothe fraction 3 in Oikawa OIK. Both DUP and OIK were almost equivalent.Oikawa et al. nevertheless taught away from the present invention sincethey mentioned that no activity was detectable in the fraction ofmolecular weight higher than 10 kDa, which is in contradiction with ourresults of FIG. 15.

[0325] Therefore, despite similarities between Balassa's and ourprocesses, the products obtained by both processes are clearly not thesame.

[0326] Amino Acid Content Comparison

[0327] The protein content of BAL, DUP and OIK samples (all of 16.5mg/mL of dry weight) was measured by the method of Lowry; results showvalues of 3.31, 0.27, and 4.15 mg/mL for BAL, DUP, and OIK sample,respectively. The ratio of protein/dry weight is very different when DUPsample is compared to BAL and OIK.

[0328] Analyses were performed to further analyze the amino acid contentof each liquid cartilage preparation. FIG. 20 illustrates the proportionof each amino acid in BAL, DUP, and OIK samples. The proportion of freeamino acids vary between each cartilage preparation: 23%, 73%, and 4% inBAL, DUP, and OIK samples, respectively. Obviously the proportion ofamino acids from protein origin also vary between each cartilagepreparation: 77%, 27%, and 86% in BAL, DUP, and OIK samples,respectively. See Table 11 for raw data: TABLE 11 Free a.a. content a.a.from protein origin Cartilage Preparation (μg/ml) (μg/ml) BAL 675 2314DUP 604  223 OIK 181 3910

[0329] The two prior art products (BAL, OIK) that have been compared toours (DUP) are yet considered as classical processes to preparecartilage extracts. The above results show that the present process(DUP) provides a product of unexpectedly good activity, as far asanti-angiogenic, anti-tumor, anti-inflammatory, and anti-collagenolyticactivities are concerned. We can assume that the present process hasindeed succeeded in providing plural hydrosoluble biologically activecomponents, or factors, in a single extract.

[0330] Direct comparison of BAL, DUP and OIK molecular profiles andprotein content demonstrated that each cartilage preparations haveparticular characteristics. Although they seem to share someconstituents it is evident that their ratio one to another is different.This is particularly important considering that DUP is anti-tumor whenadministrated orally at a dosage range below about 75 mg/kg andgradually looses this effect at higher dosage. This result suggests thatthe amount of more than a single factor is critical in the DUP liquidcartilage extract. Therefore, different cartilage preparations like BAL,DUP and OIK may show very different biological properties, since theprotein content and the proportion of each individual component variesbetween them.

[0331] Preparation of Liquid Extracts for Clinical Trials

[0332] Preliminary clinical trials were performed with shark cartilageliquid extract of the present invention. The liquid extract obtainedafter ultrafiltration was filtered of a Millipore filter of a porosityof 0.22 μm. The microbial limit of the liquid extract was controlledaccording to USP XXIII <61>standard. The liquid extract was distributedin 7 mL aliquots (about 85 mg of proteins) in aseptic flasks, frozen at−60° C. overnight and further stored at −20° C. until utilization.

[0333] Anti-Angiogenic Effect

[0334] The liquid cartilage extract was used for treatingangiogenesis-dependent diseases. Three different types representative ofangiogenesis-dependent diseases were tested in the practice in human;the first type being cancer (prostate cancer), the second type beingdermatological disorders (psoriasis and rosacea), and the third typebeing arthritis (rheumatoid arthritis and osteoarthritis). The examplesbelow will illustrate and indicate at least the anti-angiogenic activityof the liquid extract.

[0335] The results shown hereinbelow are very encouraging and are deemedpredictive of the usefulness of the cartilage extract and fractionsthereof in the treatment of all angiogenesis-dependent diseases, and notonly the ones specifically tested. Insofar as a disease has anangiogenic component, it is deemed that the cartilage extract of thepresent invention will be effective in this respect provided that itenters a composition containing an effective amount thereof and thatthis composition is in a suitable form for proper administration.Therefore, it will be appreciated that the present invention is notlimited to the following specific compositions for use in the treatmentof angiogenic diseases, since the person skilled in the art would beable to derive numerous compositions wherein choice is guided by themode of administration thereof and the targeted ill tissue. Compositionsmay be administered by different routes, e.g., topical, oral,sublingual, rectal, intravenous, intramuscular, intraocular,intraperitoneal, by diffusion, etc.

[0336] Because of the fishy taste and smell of the cartilage extract,flavoring agents or fragrances may be added or other galleniccompositions (liposomes, encapsulation, patch, etc.) can be designed tomask any unpleasant taste or smell and to encourage patient'scompliance. The term “patient” is meant to designate a human or ananimal patient. Cancer:

[0337] One patient suffering of prostate cancer has added the liquidcartilage extract to its diet and shows significant health improvementsince. An adenocarcinoma was diagnosed in 1986. At that time,radiotherapy was undertaken. In 1991, the PSA (prostatic serum antigen)level was 138 μg/L, when the normal acceptable higher limit is 4 μg/μL.The patient then underwent a completely different therapy by castrationcombined with anti-androgen therapy (EUFLEX). This treatment wasefficient during three years, after which PSA level began to rise again.Since June 1994, this patient added the liquid cartilage extract to itsdiet (daily oral dose of about 75 mg of dry weight/7 mL of extract,equivalent to about 1-1.5 mg/kg of body weight/day). The PSA levelsgradually decreased from 12 to below 4.0 μg/mL (normal limit) the lastresult being obtained in April 1996. This dose regimen would have to bemodified at will in accordance with the route of administration, thebioavailability of the active ingredients and the desired aggressivenesswith which the pathology is to be controlled. In this case, the liquidextract is probably absorbed in the gastrointestinal tract insubstantial proportions. One can rely upon the results obtained withDMBA-treated rats and inoculated mice (see above). At this time, thenon-toxicity has been verified in rat, mouse (see above examples), andmonkey (data not shown).

[0338] Oral administration of the liquid extract in DMBA-treated ratsand DA3-implanted mice suggest a dosage rate between 1 to 300 mg/kg ofbody weight, which presumably had a great contribution to the inhibitionof tumor progression and tumor vascularization in animal models.Intraperitoneal administration of the liquid extract in mice (DA3-model)demonstrated that the route of administration is important to obtain aneffective dosage in inhibiting tumor progression. This suggests that thedose rate of 1 mg/kg effective in the prostate cancer case could belowered to almost 0.01 mg/kg if a parental administration route isselected. It is therefore assumed that a dose of about 0.01 to about 200mg/kg of body weight per day is a reasonable range of median doses(ED₅₀) for treating cancer, at least partly by reducing or abolishingangiogenesis.

[0339] Several other patients added liquid cartilage extract to theirdiet (daily oral dose of about 75 mg of dry weight/7 mL of extract,equivalent to about 1-1.5 mg/kg of body weight/day) in combination withmore traditional therapies (surgery, chemotherapy, antihormonetherapy,etc.). Summaries of some medical cases are provided in the followingtable. The results suggest that combination therapy with liquidcartilage extract may increase survival rate and quality of life ofpatients suffering of solid tumors. TABLE 12 Type of cancer MedicalHistory Urinary bladder Urinary bladder 70-year old man; underwentablative surgery of several lesions (2 cm and 1.5 cm) and added liquidcartilage extract to his diet; no residual carcinoma since 09/94.Ovarian 47-year old woman; lesions of 15 cm (right), 11 adenocarcinomacm (left) and several 2 cm lesions; underwent surgery and chemotherapyin 1991; relapse treated by chemotherapy in 1992; second relapse treatedby chemotherapy in 1993; addition of liquid cartilage extract to thediet in 1994; malignant neoplasm of reduced mass since. Rhabdomyosarcoma63-year old man; infiltrating tumor of 11 cm in diameter (450 g);underwent surgery and chemotherapy; relapse treated by radiotherapy andaddition of liquid cartilage extract to the diet; tumor showing necrosedtissue and stability since. Pancreatic carcinoma 45-year old woman;pancreatic lesion (9 cm) + with liver metastases liver metastases;chemotherapy and addition of liquid cartilage extract to the diet since;tumor regressed by 80% in 1994; tumor disappeared in 1995. Mammary67-year old woman; surgery in 1978; relapse and adenocarcinoma lungmetastases (1994); Megace and addition of liquid cartilage extract tothe diet; since, partial regression of tumors in size (1.5 cm  1 cm) andnumber (12  6).

[0340] Further, the liquid cartilage extract has a direct anti-tumoreffect against melanoma cell lines. Since it also has anti-angiogenicand anti-metalloprotease activities, topical, parenteral or per ostreatment of melanoma is within the scope of this invention.

[0341] Psoriasis:

[0342] The following dermatological composition was made and tried toverify its efficacy in patients suffering of psoriasis:

[0343] EMULGADE CLB 29% (W/W)

[0344] 20× crude permeate 69.5% (W/W)

[0345] GERMABEN 11 1% (W/W), and

[0346] Lavandula Angustifolia 0.5% (W/W)

[0347] EMULGADE CLB, a mixture of stearate esters, fatty alcohols andnonionic emulsifiers (purchased from Henkel Canada Ltd.) was heated at65-70° C. under agitation. Heating was stopped while the mixture waskept under agitation. When the mixture reached a temperature of 45° C.,the essential oil Lavandula Augustifolia and the preservative agentsGERMABEN II (diazonidyl urea 30%, methylparaben 11%, propylparaben 3%and propylene glycol 56%; purchased from Sutton Laboratories, NJ,U.S.A.) were added. When the temperature of the mixture reached 30° C.,the liquid cartilage extract was added. The 20× liquid extract wasconcentrated on a membrane having a nominal molecular weight cut-offvalue of 1 kDa. The composition so obtained was a smooth non-greasycream; by varying the percentage of EMULGADE, other forms of variousviscosity dermatological compositions can be obtained, in accordancewith the manufacturer's directives (milk, lotion, ointment). Othervehicles or excipient might be used to obtain pastes, gels and any otherform of transdermal preparation.

[0348] The above formulation was given twice daily during a period oftwelve weeks to a panel of nine patients (topical application) sufferingof psoriasis that had been responsive to the conventional therapiestried but became refractory to them after a while. For this study,patients were selected for the similar and symmetrical extent ofpsoriasis on both side members. These trials were conducted in adouble-blind fashion, neither the dermatologist nor the patients knowingwhich affected side was treated with the composition containing thecartilage extract and which one was treated with a control composition.Remarkable improvement was observed in five patients whose psoriasis wasnot complicated by hyperkeratosis; for those having hyperkeratosis, theresults were moderately good. Photographs of parts of two patients,bodies are shown in FIG. 21. In FIGS. 21a and b, it is demonstrated thata patient affected by psoriasis with hyperkeratosis has neverthelessshown a very significant reduction of the erythema, associated with noprurit, after only one month of treatment. The hyperkeratosis remained,however, important. Photographs of the second patient suffering ofpsoriasis not complicated with hyperkeratosis (FIGS. 21c and d) showed amuch better improvement after a three month treatment. Since psoriasisappears to be a multifactorial disease, it is assumed that the responseof the patients depends on the importance of the involvement ofcomponents like angiogenesis and inflammation in the establishment andin the perpetuation of this condition. The anti-angiogenic activity isindeed present in our extract, as shown in DMBA-treated rats (FIG. 5),endothelial cell proliferation (FIG. 7), and EVT (FIG. 10). Theanti-inflammatory activity has also been verified (CHS model in mice).It is probable that better results might be obtained if this kind offormulation is complemented with other therapeutic agents addressing toother factors involved (keratolytic agents, additional anti-inflammatoryagents, antihistaminics, immunosuppressors, etc.).

[0349] This complementation may take the form of amending theformulation to include an effective amount of a keratolytic agent. Forexample, it could also be achieved by the separate administration ofsuch a complementary therapeutic agent, concurrently or in alternationwith the application of the present topical formulation. Furthermore,the complementary medication does not need to be administered by thesame route.

[0350] The above formulation has shown no systemic effect (the effectappears to be limited to the treated areas) and no secondary effectdespite the high proportions in liquid cartilage extract.

[0351] Per os treatment of psoriatic patients also led to an improvementof 20 to 50% in 66% of patients treated with 30 to 240 mL /day sharkcartilage extract. This confirms the oral availability of theanti-inflammatory and anti-angiogenic component of the present cartilageextract.

[0352] Rosacea:

[0353] A comprehensive review of this skin disorder by J. K. Wilkin(Arch. Dermatol. (1994) vol. 130, 359-362) indicates that rosaceadevelops as a combination of one or more of the following cutaneousstigmata: flushing, erythema, telangiectesia, facial edema, papules,pustules, ocular lesions and rhinophyma, depending on the disorder stagedevelopment.

[0354] Erythema and telangiectesia are vascular disorders, and the otherfeatures, although not of a vascular nature, may derive from a vasculardisturbance. The erythema is the first feature to be observed andrepresents an increased number of erythrocytes in a mildly inflamedvasculature. Furthermore a dermal cellulitis may appear as a result ofan extravascular fluid accumulation consequent to irritant factors. Theedema is the result of an increase extravasation along with a decreasefluid removal by lymphatic vessels. Decrease lymphatic activity appearsto be consequent to lymphatic damage occurring during cellulitis.Rhinophyma may be explained by the observation that chronic cutaneousedema is frequently followed by connective tissue hypertrophy andfibroplasia and may also be due to factor XIII expression. It has beenfurther emphasized that the elastin network that surrounds the lymphaticsystem in the skin serves two important functions. First, it is atethering that permits the lymphatic endothelium to be sensitive to thevolume of fluids in the vicinity of the lymphatic vessels, so that anyincrease of volume results in greater tension on the anchoringfilaments. Second, the elastin network provides a low-resistance pathwaythrough the intersticium along which micromolecules pass to thelymphatic vessels. Elastin degeneration due to actinic exposure isprobably a common cause of lymphatic failure in rosacea.

[0355] During inflammation, neutrophils are recruited and exacerbate therapid degradation of a variety of extracellular matrix macromolecules,especially elastin. Neutrophil elastase degrades type IV collagen in theextracellular matrix on which the integrity of the capillary wallsdepends. When lymphatic failure occurs, a sustained inflammation takesplace. When lymphatic failure does not resolve the inflammation becomesself-sustained. The plasma proteins that accumulate in the sustainedinflamed tissue appear to contribute to the fibroplasia, which underliesthe development of rhinophyma.

[0356] Telangiectesia represents the latter phase of the vascular stageof rosacea. The mechanical integrity of the dermal connective tissue isreduced, allowing a passive dilation of the vasculature. Theperivascular inflammatory cells thus infiltrate and contribute torosacea. Dilatation of both dermal blood vessels and lymphatics areprominent in rosacea. Angiogenesis may contribute to the telangiectesia.Angiogenesis depends on the space left between in a tissue whereendothelial cells can grow. Edema reduces the tissue compactness,permitting vascularization. Since lymphatic failure results in sustainedinflammatory response, the edema thus created would be one the featurethat favors angiogenesis.

[0357] As already mentioned above, it appears that rosacea is initiatedby an inflammatory reaction which does not resorb with time.

[0358] Since shark cartilage comprises a plurality of biologicalactivities such as anti-metalloprotease, anti-angiogenic, anti-tumor,anti-inflammatory and also probably (since bovine cartilage contains it)anti-elastase activities, and since it inhibits VEGF activity (it atleast inhibits the VEGF-induced extravasation), it is contemplated thatthe shark cartilage extract will have an effect on all skin diseasesinvolving one or more etiologies related to these biological activities.The shark cartilage extract of this invention comprises a plurality ofactive ingredients and as such will be effective in treating mono- aswell as in pluri-factorial diseases or disorders. The treatment ofrosacea is a typical example of such a plurifactorial disorder whereininflammation, metalloprotease activity and angiogenesis occur.

[0359] For example, a small panel of people suffering from rosacea wastreated with a shark cartilage extract topically formulated in acommercial Glaxal Base or with a non-commercial base. The liquidcartilage extract was at a concentration of 5% -10% (20× extract). Theformulation was applied twice daily for about four to twelve weeks onaffected areas of their faces. A noticeable reduction of redness and offacial blood flow was observed. Measurements with a scanning laserDoppler capillary perfusion monitor showed a 34%, 31% and 32% reductionin blood flow after 1, 2 and 3 months of treatment, respectively. Visualgrading of erythema showed improvements of 24%, 27% and 44%,respectively.

[0360] These results confirm that the cartilage extract inhibitsreddening, e.g., the erythema, of the skin typically associated withrosacea. The erythema forms as a consequence of the migration oferythiocytes into the tissue. It is likely that such a migration wasinhibited by the cartilage extract by preventing inflammation and/ormetalloprotease activity.

[0361] Arthritis:

[0362] Patients suffering of arthritis have tried on a voluntary basisone to two units of 7 mL total liquid extract per day for severalmonths. These patients saw their condition improved gradually byrecovery of joint function, diminution of pain and inflammation (up toabout 60%). Since arthritis has angiogenic and inflammatory components,the above effect can be attributed to anti-angiogenic andanti-inflammatory activities of the cartilage extract.

[0363] A pilot clinical study was then conducted by a group ofspecialists in rheumatology. Seven voluntary and enlightened subjectsaged between 39 and 60 years of age and suffering from rheumatoidarthritis enrolled in the study. Diagnosis was established based on theclassification criteria in the revised edition of the AmericanRheumatism Association's (Arnett, F. C. et al., 1988, Arthritis &Rheumatism, vol. 31, 315-325).

[0364] The treatment lasted 30 days and consisted of in ingesting adaily dose of 21 mL of liquid shark cartilage extract (12 mg/mL of dryweight). The efficacy of the treatment was determined with an articularindex for the assessment of joint tenderness (Ritchie, D. M. et al.,1968, Quarterly J. Med, New Series XXXVII, vol. 147, 393-406). The indexis based on the summation of a number of quantitative evaluations of thepain experienced by the patient when joints are subjected to firmpressure exerted over the articular margin or in some instances uponmoving the joint. The results show that four patients out of seven haveimproved when treated with the liquid cartilage extract (Table 13below), suggesting that the product may be useful in the treatment ofrheumatoid arthritis or other conditions complicated by chronicinflammation. TABLE 13 Ritchie's INDEX Patient (no) Age (years) Day 0Day 30 Improvement 1 60 30 22 Yes 2 43 8 8 No 3 52 12 12 No 4 41 15 19No 5 46 5 3 Yes 6 39 6 2 Yes 7 55 14 7 Yes

[0365] Spider Veins:

[0366] A total of sixteen panelists were recruited for the study. Thepanelists had visible but not excessive telengectasia on the face. Thepanel was divided in two groups of eight each. Group A was provided witha cholesterol liposomal base containing 5% liquid cartilage extractwhile the second group (B) was provided with the cholesterol liposomalbase alone. The products were used on the full face, twice a day forthree months. A fiber optic surface microscope was used to obtain imagesof a minimal of four sites of the face showing spider veins. The imageswere analyzed for grey values via the Zeiss Ibas Image Analyzer.Integrated Optical Density (IOD) was calculated for each site for eachpanelist. The four sites on each panelist were averaged for each timepoint.

[0367] The results show there was a 35% decrease in the IOD after fourweeks and this effect maintained for the course of the study (FIG. 22).The empty cholesterol liposomal base exhibited a background improvementof 5% and 8% after eight and twelve week use, respectively.

[0368] Peri-Orbital Dark Circles:

[0369] Skin coloration is not entirely due to the presence or absence ofmelanin, but also blood supply and plasma contents. When the blood flowis sluggish and greater amounts of oxygen are removed for metabolism,the skin appears bluish in color. These color differences areexaggerated in the eye area because of the thinness of the skin (Oresajoet al. (1987) Cosmetics Toiletries 102: 29-34). Vascular changes in thesepta that are present under the eye can also exacerbate the appearanceof dark circles. Dark circles around the eyes also appear due to fatdeposition, edema under the eyelids and leakage of blood vessels aroundthe eye area. These symptoms appear to be inflammation and angiogenesisrelated. Clinical study was designed to evaluate the effect of sharkcartilage liquid extract on controlling angiogenesis around the eye areathereby reducing the appearance of peri-orbital dark circles.

[0370] A total of eighteen female volunteers between the age of 18-65participated in the study. The panelists exhibited distinct dark circlesaround the eyes. All panelists were normal in health with no evidence ofacute or chronic diseases including dermatological or ophthalmologicproblems.

[0371] Subjects exhibiting current sunburn, rashes, scratches, burnmarks, etc., which might interfere with evaluation of test results wereexcluded from the study. Pregnant or lactating females were alsoexcluded. The test site was devoid of warts, moles, sunburn, suntan,scars, and active dermal lesions observed upon observation.

[0372] The panel was divided in two groups, ten in group A and eight ingroup B, each corresponding to the vehicle containing 5% liquidcartilage extract or the vehicle alone, respectively. The panelists wereprovided with enough product to be applied around the eye area at leasttwice a day for twelve weeks. Measurements were obtained at baseline,and after four, eight, and twelve weeks. At each visit photographs wereobtained and analyzed via Image Analysis.

[0373] Photographs were analyzed for the gray values that depictdarkness/lightness of skin. It is clear that the group treated withliquid cartilage extract exhibited a good increase in gray values (whichdepicts lightening of dark coloration). After four, eight, and twelveweeks there was 11%, 21%, and 14% lightening of the skin under the eyearea of the group treated with the liquid cartilage extract. The grouptreated with the vehicle alone did not show any change (FIG. 23).

[0374] Varicose Veins:

[0375] A total of twenty panelists completed the study. The panelistshad visible but not excessive telangiectasia on the legs. The panel wasdivided in two groups, Group A (n=9) was provided a liquid cartilageextract containing cream while Group B (n=11) was provided a vehiclecream alone to be used on the full legs, twice a day for three months. Afiber optic microscope was used to obtain images of 2-4 sites of thelegs showing varicose veins. The images were analyzed for grey valuesvia the Zeiss Ibas analyzer. Integrated Optical Density (IOD) wascalculated for each site for each panelist. All the sites on eachpanelist were averaged for each time point.

[0376] Results are illustrated on FIG. 24. There was 21%, 17% and 26%decrease in the IOD after four, eight and twelve weeks of use,respectively. The control vehicle exhibited a background improvement of5%, 0% and 0% after four, eight and twelve weeks of use, respectively.

[0377] Other potential clinical and veterinary applications:

[0378] Ophthalmology:

[0379] A decrease in vision or blindness can be caused by a number ofconditions characterized by abnormal blood vessel growth orneovascularization. These include corneal neovascularization (caused bychemical or physical irritation), corneal infection, corneal graftrejection, neovascular glaucoma, macular degeneration, herpes viruskeratitis, and diabetic retinopathy. The liquid cartilage extract couldact upon these clinical conditions by inhibiting the formation of newblood vessels, and by reducing telangiectasis and inflammation.

[0380] Wound Repair:

[0381] Wound repair involves a complex interaction between cells,biochemical mediators, extracellular matrix molecules, and the cellularmicroenvironment. After full-thickness wounding, granulation tissue(fibroblasts, capillaries, and inflammatory cells) first grows from thewound edge in a characteristic sequence. Fibroblasts begin to migrateinto the wound space from connective tissue at the wound edge within 24hours. As they move, fibroblasts produce matrix molecules (collagen andglycosaminoglycans), which form an extracellular matrix. The firstcapillary buds can be seen in the perfused microcirculation at the woundedge as early as 18 hours after wounding. These buds grow into the woundspace and provide the new capillary network for the wound connectivetissue. Fibroblast proliferation and migration and capillary growthcontinue as a unit until the wound space is completely filled with newtissue. Some wound repair conditions that are complicated byoverexpression of granulation factors, such as hypertrophic scarring andthe healing of the skin of badly burned person, could benefit of theadministration of liquid cartilage extract (orally or topically) sincedecreasing the angiogenic process would slow down the process of woundhealing and repress overexpression of granulation factors.

[0382] Papulosquamous Skin Disease:

[0383] The beneficial effect of liquid cartilage extract on psoriasiticlesions suggests that other diseases having common characteristics couldalso profit of local or systemic administration of the liquid extract.The papulosquamous skin diseases are characterized by red to violaceouspapules and plaques that result from thickening of the epidermis and/orunderlying dermal inflammation and include psoriasis, Reiter's syndrome,pityriasis rosea, lichen planus, pityriasis rubra pilaris, secondarysyphilis, mycosis fungoides, and ichthyosiform eruptions.

[0384] Alopecia:

[0385] The ligature of small lateral arteries driving blood flow to thescalp has been successful to prevent hair loss caused by androgenoverexposure. Local application of liquid cartilage extract on someregion of the scalp could prevent hair loss by decreasing the vascularnetwork and consequently the exposure to hormones.

[0386] Veterinary Applications:

[0387] Solid and/or liquid cartilage extracts may be administered toanimals for the same therapeutic and cosmetic applications that havebeen described for humans.

[0388] Most, if not all, of the above-mentioned diseases, which havebeen classified as representative of angiogenic-related diseases, appearto have an inflammatory etiological component as well as a matrixmetalloprotease etiological component. There is an increasing body ofliterature that shows that inflammation is one the early events that areinvolved in signaling a danger to the body. Inflammation initiates otherevents like blood cell recruitment to the endangered site, which cellsare then activated and secrete cytokines. Matrix metalloproteases aresecreted in order to break the extracellular matrix down and tofacilitate other cell recruitment. Inflammation is usually stopped whenthe danger has disappeared and the tissue has returned to normalfunction. However, in some cases, inflammation is self-sustained andleads to chronic disease which tends to aggravate with time. A majorityof diseases or disorders is plurifactorial so that it is almostimpossible to draw a line between angiogenesis-, inflammation-,metalloprotease-, and cell proliferation-related diseases. In manydocumented cases, namely rosacea, angiogenesis appears at a late stage,wherein the disorder aggravates as a consequence of a lack of treatmentthat would have controlled inflammation at an earlier stage.

[0389] Non-Anti-Angiogenic Effect

[0390] Acne:

[0391] Even though acne is not generally considered a disease ordisorder having an angiogenic component, it was nevertheless tempting totest the liquid cartilage extract in patients so affected on the basisthat the liquid extract is also anti-inflammatory. For experimenting thecartilage extract in patients affected by acne, the following gelformulation was made:

[0392] CARBOPOL 1.2%

[0393] Purified water 77.2%

[0394] NaOH 0.3%

[0395] PHENOXETOL 0.3%

[0396] TWEEN 80 0.3%

[0397] 2× Liquid cartilage extract 20.0%

[0398] 40× Aloe extract 0.5%

[0399] The liquid cartilage extract contains 9-12 mg/mL of dry weightand was concentrated on a membrane of NMWCO of 1 kDa. This formulationshows a remarkable improvement of the aspect of the skin of patientsaffected by more or less severe forms of acne (inflammatory acne andcystic acne). FIG. 25 shows the significant improvement of the conditionof a patient suffering of acnea when treated with the topical liquidextract containing vehicle during twelve weeks.

[0400] These results may be due to an anti-angiogenic effect (thusrevealing an angiogenic component in acne), or they may be due to thepresence of active ingredients in the liquid extract that have an effectother than anti-angiogenic (an anti-inflammatory effect, for example).All the results obtained in the above clinical trial show the greatpotential of the cartilage liquid extract in the treatment ofangiogenesis-dependent and/or inflammatory diseases. The amount ofcartilage extract as well as the formulation thereof may be varied atwill to fulfil specific needs.

[0401] One can note that, on a protein content basis, the topical andall other compositions may contain a wide range of doses of thecartilage extract. Among the three specific categories of cases tested,very different dosages and/or formulations have been used. Skinirritancy:

[0402] Since angiogenesis is often associated to inflammation innumerous diseases, it would be desirable to assign each activityseparately in the cartilage extract. In this regard, a skin irritationmodel wherein no angiogenesis is suspected to occur has been chosen totest the extract for its anti-inflammatory and soothing activity. Ninevolunteers with a history of skin sensitivity to balsam of Peru werechosen for the study. The test compounds were as follows:

[0403] 1. IX Shark cartilage 50% in D-MEM media

[0404] 2. IX Shark cartilage 20% in D-MEM media

[0405] 3. IX Shark cartilage 10% in D-MEM media

[0406] 4. Cola nitida (Indena) 10% Hydro-alcohol 1:1.

[0407] The four test compounds were applied on the ventral forearms ofthe panelists. The material was allowed to absorb for twenty minutes andthen balsam of Peru, an irritant, was applied on the test sites. Skinirritation was measured in terms of increase in skin redness. The degreeof redness was measured with a Minolta Chromameter and compared with thepositive and negative controls. The positive control was the color ofskin treated with balsam of Peru alone and the negative control was askin site treated with cola solution and challenged like the testproducts. Statistical significance was calculated via two tailedprobability T-test. FIG. 26 shows that cola at 10% was 70% active. Sharkcartilage was 58% and 60% as anti-irritant at 20% and 10%concentrations, respectively. There was no dose-response effect. Theseresults suggest that the cartilage extract contains anti-inflammatoryand soothing activity which is remote from an anti-angiogenic effect.

[0408] Many types of skin irritants are known and include, for example,chemicals, physical abrasion, U.V. radiation, allergens, or infectiousagents (fungi, viruses and bacteria). The anti-inflammatory activity ofthe present cartilage extract counteracts the action of these variousirritants and is therefore useful in treating disorders or diseasesrelated to them.

[0409] Cancer:

[0410] A 53 year old female patient was diagnosed as having a large cellnon-Hodgkin's lymphoma of the B type. CAT scan analysis revealedadenopathies around the carotid and the jugular vein (2.5 cm indiameter) and a voluminous adenopathy over the right renal hilus. Thepatient refused chemotherapy and added the liquid cartilage extract toher diet (October 1993)(daily oral dose of about 75 mg of dry weight/7mL of extract, equivalent to about 1-1.5 mg/kg of body weight/day).Three months later (January 1994) CAT scan analysis revealedadenopathies in the neck completely resorbed. By November 1994 theabdominal adenopathy has decreased in volume by 75%.

[0411] This result suggests that some non solid cancers may also respondto the anti-tumor activity of the liquid cartilage extract.

[0412] Barrier Protection of the Skin:

[0413] A panel of six healthy volunteers participated in the study. Thepanelists received a cream containing the liquid cartilage extract to beapplied on the right forearm and the vehicle only to be applied on theleft forearm, twice a day for four weeks.

[0414] The panelists were female, ages 21-45, with no evidence of acuteor chronic disease including dermatological or ophthalmologic problems.Subjects exhibiting current sunburn, rashes, burn marks, etc., whichmight interfere with evaluation of test results, were excluded from thestudy. The test site was devoid of warts, nevi, moles, sunburn, suntan,scars and active dermal lesions observed upon examination. On the day ofthe test, the panelists were instructed to refrain from using anylotions, creams or other products on the face. During the course ofmeasurements, the panelists were equilibrated for at least 30 minutesprior to testing in a controlled environment of 20-22° C. temperatureand 40% relative humidity.

[0415] The test site was the right and left volar forearms. A small area(3.5 cm×7 cm) was marked on each arm and basal transepidermal water loss(TEWL) measurements were obtained from three sites within this area(Pinnagoda et al. (1990) Contact Dermatitis 22: 164-178; Grove (1994) inThe effects of aging in oral mucosa and skin, Ed. Squier & Hill, CRCPress, pp 124-125).

[0416] A sticky (Tuck) tape was used to cover the test area and, after afirm stroke in both directions, the tape was peeled off (Elias (1993) J.Invest. Dermatol. 80: 044s-049s). A total of 5 strippings were obtained.TEWL was recorded again. Strippings followed by TEWL measurements werecontinued in groups of 5. The strippings were stopped when the TEWLapproached 18 G/M²/Hr. TEWL was measured again at the end of the laststripping sequence. The number of strippings required to damage the skinbarrier was calculated by noting the number of maximum strippings foreach arm, at each time point that exhibited a TEWL of 18 G/M²/Hr ormore. The results were analyzed for statistical significance betweentreatment at various time points versus baseline using the one tailedrank coefficient Z test.

[0417] The vehicle treated arm did not appear to exhibit muchimprovement. Only 26% and 21% more stripping was required to damage skinafter 2 week and 4 week treatments, respectively. There was asignificant improvement (p<0.05) in barrier condition of each of thepanelists after treatment with liquid cartilage extract product for 2and 4 weeks when 60% and 55% more strippings were required to disruptskin barrier (FIG. 27).

[0418] Therefore, the liquid cartilage extract has proven to be usefulin strengthening skin barrier against physical damage. Without beingbound to any theory, it is believed that this effect is due to theanti-inflammatory and anti-metalloprotease activities of the cartilageextract.

[0419] Eczema:

[0420] The liquid cartilage extract was tested in beauty salon on thebasis of its ability to decrease the inflammatory lesions caused byeczema. The beautician applying the liquid extract-containing creamsuffers since many years of chronic eczema in her hands. Interestingly,the uses of the cartilage-containing cream decreased significantly theexpression of eczema in her hands. She is now using successfully thecartilage-containing cream to prevent the expression of eczema.

[0421] Warts:

[0422] A 36 years old female with a history of plantar warts was treatedby a dermatologist for almost three years to control wart progressionand associated pain. Among the treatments there was liquid Nitrogen,Salicylic acid (40%), Anaerobia, Nitric acid, and sulfuric acid. Thesetreatment were generally every week for a duration of three months andthe results were almost nil. In March 1996 she applied daily (5 minutes)the shark cartilage liquid extract directly on the warts; two weekslater a pink zone of new epidermis formed around the warts; thefollowing week warts were gone. Therefore, this result suggests that theliquid cartilage extract may help in the treatment of warts,particularly by its anti-metalloprotease activity. Warts have adecreased chance of remaining implanted in the skin.

[0423] Other Potential Clinical and Veterinary Applications:

[0424] Graft Rejection:

[0425] Inflammation is one of the major factor involved in the earlymortality of transplanted cells. Therefore, tissue graft could benefitfrom the anti-inflammatory components present in our shark cartilageliquid extract. Furthermore, an anti-metalloprotease activity should beresponsible for the inhibition of recruitment of inflammatory and immunecells to the transplantation site.

[0426] Multiple Sclerosis:

[0427] The cause of multiple sclerosis are unknown. The tissue responsehas features of an immunopathologic process, with perivenularmononuclear cell infiltration and absence of any overt histopathologicevidence of an infection. Matrix metalloproteinases are importantfactors involved in inflammatory response. Since liquid cartilageextract is a powerful inhibitor of matrix metalloproteinases it may beuseful in the treatment of multiple sclerosis.

[0428] Fibrosis:

[0429] Current concepts suggest that fibrosis resembles normal woundhealing, but fail to terminate, leading to the replacement of normaltissue with scar. Most fibrotic reactions appear secondary to trauma,infection, inflammation or, for unknown reasons, may have a geneticcomponent. Typically TGF-β is overproduced and induces the proliferationof fibroblastic cells and the overproduction of collagen. Since anexcessive deposition of collagen is the hallmark of fibrosis, we suggestthat liquid cartilage extract which can delay the formation ofgranulation tissue could have long term benefits in suppressing fibroticreactions.

[0430] Inflammatory Bowel Disease:

[0431] The etiology of inflammatory bowel disease is unknown, butabnormal intestinal immunity is involved in the pathogenesis of Crohn'sdisease and ulcerative colitis. Mucosal mononuclear cells displayaltered antibody production, proliferation, cytotoxicity and cytokinesynthesis (FGF, PDGF, EGF, TNF). Liquid cartilage extract has shownanti-inflammatory activity and then oral administration may prove to behelpful in the therapeutical treatment of inflammatory bowel disease.

[0432] Heart Diseases:

[0433] Endothelial dysfunction of coronary resistance vessels canaccount for abnormalities of the coronary microvasculature and possiblymyocardial ischemia and chest pain. At a cellular level, endothelialdysfunction is associated with reduced expression of nitric oxide (NO),an endothelium-derived relaxing factor. NO synthesis allows the vascularsystem to maintain a state of vasodilatation thereby regulating arterialpressure. A deficit in endogenous synthesis of NO contributes to suchconditions as arterial hypertension, pulmonary hypertension and heartdisease. We have preliminary results from cultured endothelial cellsthat the liquid cartilage extract increases NO production. The liquidextract might therefore, through NO, prove to be helpful in some heartdisease conditions as well as in pediatric patients with congenitalheart disease complicated by pulmonary artery hypertension.

[0434] Moreover, liquid cartilage extract may help to decreaseinflammation-associated complications in atherosclerosis via itsanti-inflammatory and anti-MMP components.

[0435] Scleroderma:

[0436] Scleroderma (Hard skin) is an uncommon disease marked byincreases in fibrotic connective tissue of skin and often of visceralorgans as well. It often appears as a hyperkeratinization of localizedskin patches. Hyperkeratinization is a cellular process in whichkeratinocytes of the skin fully differentiate and accumulate rigidkeratin fibers. This skin condition might lead to limited joint mobilityif skin in a periarticular area is affected. When added to anexperimental system in which keratinocyte differentiation is encouraged,the liquid cartilage extract partially prevents the process ofdifferentiation, or keratinization. Therefore, the liquid extract mightbe beneficial for such skin conditions by preventing theoveraccumulation of fully differentiated keratinocytes.

[0437] Veterinary Applications:

[0438] Solid and/or liquid cartilage extracts may be administered toanimals for the same therapeutical and cosmetic applications that havebeen described for humans.

[0439] Cosmetic Applications and Compositions:

[0440] The above tests and trials have shown that the cartilage extractof this invention may find numerous medical applications. Among thediverse activities recovered in this extract, anti-angiogenic,anti-collagenolytic, anti-inflammatory and the inhibitory effect onPKC-induced differentiation are particularly desirable in cosmeticapplications. Since the cartilage extract of the present invention hasshown an antagonist effect of PKC-mediated cellular events, and sincesuch antagonist effect is suggested in the art as one improving the skinbarrier function, a method for improving the barrier function inmammalian skin which comprises the step of applying to the skin acomposition which comprises the cartilage extract and a pharmaceuticallyacceptable carrier, and such a composition are within the scope of thisinvention. Other or similar compositions can also be conceived to beused in a method for soothing skin or for reducing inflammation inmammalian skin. Inflammation can be caused by various agents such aschemical irritant, physical abrasion and exposure to ultravioletradiation. Compositions and methods for inhibiting collagenase in skinare also contemplated. Collagenase and inflammation are linked topremature aging (degradation of collagen), and therefore the antagonistactivities recovered in the cartilage extract could also be put tocontribution in compositions and methods for retarding premature aging,and for regulating wrinkles or atrophy in mammalian skin. As causes ofwrinkles or atrophy are listed, by way of examples, age, exposure toultraviolet radiation or to environmental pollutant. Topicalcompositions may comprise an effective amount of shark cartilage, to bedetermined for each specific application. In general, these compositionsmay contain from about 0.1 to about 75 weight percent of a liquidcartilage extract and from about 25 to 99.9 weight percent of apharmaceutically acceptable vehicle. These compositions may contain ananti-oxidant, such as an agent which prevents the formation of lipidperoxides in skin. Examples of such an anti-oxidant are tocopherol,tocopherol derivatives, ascorbic acid, ascorbic acid derivatives andBHT. The compositions can be complemented with anti-inflammatory agentslike a phospholipase A2 inhibitor or the botanically-derivedanti-irritants cola and green tea extract. Topical compositions may takediverse forms such as solutions, suspensions, lotions, tinctures, gels,creams, sprays, emulsions, sticks, ointments or liposomes (at least aportion of the liquid cartilage extract being present in liposomes).Other cosmetic applications include dark circle around the eyes and skinbarrier function.

[0441] The process of the present invention has been demonstrated as onethat provides for the production of cartilage extracts of clinicalvalue. The shark cartilage extracts produced by this novel processcomprises a multiplicity of activities that are recovered in goodyields. The cartilage extracts, particularly the liquid extract andfractions thereof have a great potential since they are non-toxic tonormal cells while they are effective in a large variety of diseases orconditions.

[0442] For all predicted applications (from ophthalmic drops todermatological and cancer drug formulations), it is presumed that aminimal final protein concentration of the total liquid extract could bevery low (from about 0.01 mg/ml). This lower range of doses depends onthe accessibility and on the permeation of the active ingredients to thesite of action as well as on the efficient capture of these ingredientsand the sensitivity or response of the tissue to angiogenic inhibitors.The highest limit of the final protein concentration in formulations forsome applications is not currently known. The highest finalconcentrations tried were a topical administration of about 9 mg/mL ofproteins in the formulation for the psoriasis cases and an oraladministration of about 12 mg/mL in the dose unit of 7 mL administereddaily in the cancer cases and 21 mL in the arthritis trial.

[0443] The shark cartilage liquid extract may lose some of itsactivities when lyophilized. However, the addition of stabilizers orprotective agents as known in the art prior to lyophilization maypreserve sensitive activities and make possible the administration ofhigher doses of the cartilage extract in the dry state.

[0444] Materials Used in Preparation/Use of Invention:

[0445] Coolers

[0446] Surgical instruments

[0447] Meat chopper

[0448] Plastic bags

[0449] Industrial blender (Waring 3-speed blender bought from FisherScientific)

[0450] A system of purification of water (inverse osmosis and 0.1 Emfiltration; Continental Water System, model PRE 2202, serial number91089, Modulab Bioscience RQ/Polishing System bought from FisherScientific, Montreal, Quebec). This system provides an apyrogenic waterof high quality.

[0451] A precision balance Mettler, series AE bought from FisherScientific

[0452] Centrifuge Sorvall RC-285 bought from DuPont Canada

[0453] Centrifuge CEPA

[0454] Nylon pocket of a porosity of 1 μM

[0455] An autoclave (automatic vapor sterilizer Sanyo, model MAC 350P)

[0456] Nalgene 500 mL containers sterilized at 132° C. for 10 minutesand dried for 35 minutes

[0457] Conical filters of 24 μm porosity Whatman Reeve Angel

[0458] Ultrafiltration column (Molecular weight cut-off: 500 kDa, 1 kDa,and 0.1 kDa when applicable; Surface: 25 square feet; Flow: 130L/minute; Inlet pressure: 30 psi; Outlet pressure: 5 psi; bought fromKoch Membrane Systems Inc., Wilmington, Mass., USA)

[0459] Sanitary centrifuge pump (Monarch industries, model ACE-S 100,type A) for providing a 130 L/minute flow

[0460] sterile hot (laminar flow hot NuAire bought from Ingram & Bell)

[0461] Millipack-60 0.22 μm sterile filters

[0462] Sterile clear or amber glass bottles

[0463] Concentrator DC-10 Amicon

[0464] Rotofor BioRad 170-2950

[0465] Amicon filters SIOY10, SIOY30 and SIOY100 of cut-off values of10, 30 and 100 kDa, respectively

[0466] FPLC Pharmacia 216007 (computer Pharmacia 216014)

[0467] Hilstand S-300 26 mm/60 cm (Pharmacia)

[0468] Superose S-12 10 mm/30 cm (Pharmacia)

[0469] Lyophilizer Labconco 10273 A

[0470] This invention has been described hereinabove, and it should beappreciated that it would be well within the ability and the knowledgeof the person skilled in the art, without departing from the teachingsof this disclosure, to bring modifications by replacing some elements ofthis invention as practiced by their equivalents, which would achievethe same goal thereof. These obvious variations are deemed covered bythis application.

1. A method of preparing a fractionated cartilage extract comprisingwater soluble biologically active components the majority of which havea molecular weight of less than about 500 kDa, the method comprising thestep of: first fractionating a crude cartilage extract comprising watersoluble biologically active components obtained from cartilage materialsuch that a major portion of the biologically active components having amolecular weight of greater than about 500 kDa are separated from amajor portion of biologically active components having a molecularweight of less than about 500 kDa to form a first fractionated cartilageextract.
 2. The method of claim 1, wherein said first fractionating stepemploys at least one of a first separation medium having a nominalmolecular weight cutoff (NMWCO) of about 500 kDa, a firstchromatographic medium, and a first electrophoretic medium.
 3. Themethod of claim 2, wherein said first fractionating step is a filtrationstep and said first separation medium is a filtration membrane.
 4. Themethod of claim 1 further comprising the earlier step of: treating aparticle size-reduced cartilage solid with an aqueous solution for aperiod of time and at a temperature sufficient to extract said watersoluble biologically active components from said particle size-reducedcartilage solid.
 5. The method of claim 4 further comprising the earlierstep of: reducing the particle size of a cartilage material bymechanical means to form a particle size-reduced cartilage solid.
 6. Themethod of claim 5, wherein said particle size-reduced cartilage solidhas an average particle size of less than about 500 μm.
 7. The method ofclaim 5, wherein said treating step and said reducing step is conductedin an aqueous solution.
 8. The method of claim 7, wherein said treatingstep and said reducing step are conducted in the same aqueous solution.9. The method of claim 7, wherein said treating step is conducted duringand after said reducing step.
 10. The method of claim 5, wherein saidreducing step employs homogenization of said cartilage material.
 11. Themethod of claim 1 further comprising the step of removing a majorportion of water present in said first fractionated cartilage extract.12. The method of claim 1, wherein said cartilage material is sharkcartilage.
 13. The method of claim 12, wherein the cartilage extractpossesses anti-angiogenic and anti-tumor activities.
 14. The method ofclaim 1, wherein one or more of said water soluble biologically activecomponents comprises a protein.
 15. The method of claim 4 furthercomprising the step of: separating said particle size-reduced cartilagesolid from said aqueous solution after said treating step.
 16. Themethod of claim 15, wherein said separating step employs at least one offiltration or centrifugation.
 17. The method of claim 4, wherein saidaqueous solution is a non-denaturing aqueous solution.
 18. The method ofclaim 13, wherein said cartilage extract induces a decrease in tumorsize.
 19. The method of any one of claims 1-18 further comprising thestep of: removing a major portion of water present in said firstfractionated cartilage extract.
 20. The method of claim 1, furthercomprising the step of: second fractionating said first fractionatedcartilage extract to remove a major portion of water solublebiologically active components having a molecular weight less than about0.1 kDa and to form a second fractionated cartilage extract comprisingwater soluble biologically active components having a molecular weightbetween about 0.1 kDa to about 500 kDa.
 21. The method of claim 20,wherein said first and second fractionating steps are conductedconcurrently or sequentially.
 22. The method of claim 20, wherein saidsecond fractionating step employs a second separation medium having anominal molecular weight cutoff (NMWCO) of about 0.1 kDa, a secondchromatographic medium or a second electrophoretic medium.
 23. Themethod of claim 22, wherein said second fractionating step is afiltration step and said second separation medium is a filtrationmembrane.
 24. The method of claim 1 further comprising the step of:second fractionating said first fractionated cartilage extract to removea major portion of water soluble biologically active components having amolecular weight less than about 1 kDa and to form a second fractionatedcartilage extract comprising water soluble biologically activecomponents having a molecular weight between about 1 kDa to about 500kDa.
 25. The method of claim 24, wherein said first and secondfractionating steps are conducted concurrently or sequentially.
 26. Themethod of claim 24, wherein said second fractionating step employs asecond separation medium having a normal molecular weight cutoff (NMWCO)of about 1 kDa, a second chromatographic medium or a secondelectrophoretic medium.
 27. The method of claim 26, wherein said secondfractionating step is a filtration step and said second separationmedium is a filtration membrane.
 28. A method for treating a skindisease or disorder having an etiology related to angiogenesis, themethod comprising the step of applying to the skin of a patient in needof such treatment a topical composition comprising a therapeuticallyeffective amount of a shark cartilage extract comprising one or morebiologically active components, a majority of which have a molecularweight less than about 500 kDa.
 29. The method of claim 28, wherein themethod is a method of reducing telangiectasia.
 30. The method of claim29, wherein the telangiectasia is at least one of varicose veins and ofspider veins.
 31. The method of claim 29, wherein the method is a methodof reducing periorbital dark circles.
 32. The method of claim 29,wherein the method is a method of reducing the redness caused byrosacea.
 33. A method of treating warts in mammalian skin, said methodcomprising the step of applying to the skin of a patient in need of suchtreatment a topical composition comprising a therapeutically effectiveamount of a shark cartilage extract comprising one or more biologicallyactive components, a majority of which have a molecular weight less thanabout 500 kDa.
 34. A method of treating a papulosquamous skin disease ordisorder, said method comprising the step of applying to the skin of apatient in need of such treatment a topical composition comprising atherapeutically effective amount of a shark cartilage extract comprisingone or more biologically active components, a majority of which have amolecular weight less than about 500 kDa.
 35. The method according toclaim 34, wherein the disease or disorder is selected from the groupconsisting of Reiter's Syndrome, pityriasis rosea, lichen planus,pityriasis rubra pilaris, secondary syphilis, mycosis fungoides, andichthyosiform eruptions.
 36. A method of promoting wound repair in amammal, the method comprising the step of applying to the skin of apatient in need of such treatment a topical composition comprising atherapeutically effective amount of a shark cartilage extract comprisingone or more biologically active components, a majority of which have amolecular weight less than about 500 kDa.
 37. A method of treating aninflammatory or angiogenic ophthalmic disease or disorder in a mammal,the method comprising the step of applying to the eye of the mammal inneed of such treatment an ophthalmic composition comprising atherapeutically effective amount of a shark cartilage extract comprisingone or more biologically active components, a majority of which have amolecular weight less than about 500 kDa.
 38. The method according toclaim 37, wherein the ophthalmic disease or disorder is selected fromthe group consisting of corneal neovascularization, corneal infection,neovascular glaucoma, macular degeneration, and diabetic retinopathy.39. A method of treating a disease or disorder selected from the groupconsisting of hypertrophic scar, alopecia, multiple sclerosis, fibrosis,inflammatory bowel disease, scleroderma, vasoconstrictive diseases,herpes virus keratitis, and organ graft rejection, the method comprisingthe step of administering to a patient in need of such treatment acomposition comprising a therapeutically effective amount of a sharkcartilage extract comprising one more biologically active components, amajority of which have a molecular weight less than about 500 kDa.
 40. Amethod of treating a disease or disorder having an etiology related toany one of tumour proliferation, angiogenesis, metalloprotease activityand inflammation, the method comprising the step of administering atherapeutically effective amount of a shark cartilage extract comprisingwater soluble biologically active components, a majority of which have amolecular weight lower than about 500 kDa, the extract being obtainedfrom a process comprising the step of: fractionating a crude cartilageextract comprising water soluble biologically active components obtainedfrom cartilage material, such that a major portion of the biologicallyactive components having a molecular weight greater than about 500 kDais separated from a major portion of biologically active componentshaving a molecular weight of less than about 500 kDa to form the sharkcartilage extract.
 41. The method of claim 40, wherein the disease ordisorder is one affecting skin or mucosae.
 42. The method of claim 40,wherein the method is one or more of: a method for reducing inflammationin mammalian skin; a method for inhibiting metalloprotease activity inmammalian skin; a method for inhibiting endothelial cell proliferationin mammalian skin; a method for inhibiting cancer cell proliferation inmammalian skin; a method for enhancing skin barrier function inmammalian skin; a method for regulating wrinkles and atrophy inmammalian skin; a method for retarding premature aging in mammalianskin; a method for soothing irritation in mammalian skin; a method forinhibiting activated-keratinocyte differentiation; and a method fordecreasing the expression of eczema or acne in mammalian skin.
 43. Themethod of claim 42, wherein said inflammation is caused by a chemicalirritant, a physical abrasion, U.V. radiation, an allergen or aninfectious agent.
 44. The method of any one of claims 28, 33, 34, 36,37, and 39, wherein the shark cartilage extract has been prepared by aprocess comprising the step of: first fractionating a crude cartilageextract comprising water soluble biologically active components obtainedfrom shark cartilage material such that a major portion of thebiologically active components having a molecular weight greater thanabout 500 kDa is separated from a major portion of biologically activecomponents having a molecular weight of less than about 500 kDa to formthe shark cartilage extract.
 45. The method of claim 44 furthercomprising the earlier step of: treating a particle size-reducedcartilage solid with an aqueous solution for a period of time and at atemperature sufficient to extract said water soluble biologically activecomponents from the particle size-reduced cartilage solid.
 46. Themethod of claim 45 further comprising the earlier step of: reducing theparticle size-reduced cartilage solid.
 47. The method of claim 44further comprising the later step of: removing a major portion of waterpresent in said first fractionated cartilage extract.
 48. The method ofclaim 44, further comprising the step of: second fractionating saidfirst fractionated cartilage extract to separate and remove a majorportion of water soluble biologically active components having amolecular weight less than about 0.1 kDa to form a second fractionatedcartilage extract comprising water soluble biologically activecomponents having a molecular weight between about 0.1 kDa to about 500kDa.
 49. The method of claim 44 further comprising the step of: secondfractionating said first fractionated cartilage extract to separate andremove a major portion of water soluble biologically active componentshaving a molecular weight less than about 1 kDa and to form a secondfractionated cartilage extract comprising water soluble biologicallyactive components having a molecular weight between about 1 kDa to about500 kDa.
 50. A composition comprising an effective amount of a sharkcartilage extract comprising water soluble biologically activecomponents, a majority of which have a molecular weight lower than 500kDa, the extract being prepared by a process comprising the step of:first fractionating a crude cartilage extract comprising water solublebiologically active components obtained from cartilage material suchthat a major portion of the biologically active components having amolecular weight greater than about 500 kDa is separated from a majorportion of biologically active components having a molecular weight ofless than about 500 kDa to form said shark cartilage extract, and apharmaceutically acceptable carrier.
 51. The composition of claim 50,wherein the composition is one of a topical, ophthalmic and cosmeticcomposition.
 52. The composition of claim 50, wherein the composition isone of an ointment, cream, emulsion, suspension, gel, liquid, tincture,spray, stick, liposome, ophthalmic drop and lotion.
 53. The compositionof claim 50 further comprising at least one of: an antioxidant, ananti-inflammatory agent, an anti-irritant, a keratinolytic agent, asurface active agent, a preservative, a stabilizer, a synthetic polymer,a buffer, a cream base, an ointment base, and a salt.
 54. Thecomposition of claim 50, wherein the process of preparing the cartilageextract further comprises the step of concentrating said shark cartilageextract.
 55. The composition of claim 54, wherein the step ofconcentrating comprises one or more of: second fractionating the sharkcartilage extract to remove a major portion of water and water solublecomponents having a molecular weight less than about 0.1 kDa; secondfractionating the shark cartilage extract to remove a major portion ofwater and water soluble components having a molecular weight less thanabout 1 kDa; and lyophilizing or evaporating a major portion of theliquid from said shark cartilage extract.
 56. A process for preparing acartilage extract comprising one or more antiangiogenic water solublecomponents, said process comprising the steps of: a) first fractionatinga crude cartilage extract comprising water soluble biologically activecomponents obtained from cartilage material such that a major portion ofthe biologically active components having a molecular weight greaterthan about 500 kDa is separated and removed from a major portion ofbiologically active components having a molecular weight of less thanabout 500 kDa to form a first fractionated extract; b) secondfractionating said first fractionated extract such that a major portionof the biologically active components having a molecular weight lowerthan about 10 kDa is separated and removed from a major portion of thebiologically active components having a molecular weight comprisedbetween about 10 to 500 kDa, to form a second fractionated extract; andc) third fractionating said second fractionated extract on an anionexchange chromatography medium to recover a third fractionated extractwhich elutes in a NaCl concentration gradient and has an antiangiogenicactivity to form said cartilage extract.
 57. The process of claim 56,wherein the third fractionated extract elutes at 0.8 to 1 M NaCl and thechromatography medium is Mono-Q.
 58. The process of claim 57, whereinsaid cartilage material is obtained from shark.
 59. A cartilage extractprepared according to the process of claim
 58. 60. An antiangiogeniccomposition comprising the cartilage extract of claim 59 and apharmaceutically acceptable carrier.
 61. A matrix metalloproteaseinhibitor isolated from shark cartilage, which has an apparent molecularweight of about 31 KDa and which cross-reacts with anti-TIMP antibodies.62. The matrix metalloprotease inhibitor of claim 61 which is effectiveagainst MMP-2, MMP-9 and MMP-12 metalloproteases.